microRNA Biomarkers for Human Breast and Lung Cancer

ABSTRACT

The present invention relates to novel molecular markers for diagnosis and classification of human breast cancer and lung cancer.

This application is a continuation of U.S. Ser. No. 12/436,576, filedMay 6, 2009, which is a continuation-in-part of U.S. Ser. No.11/730,570, filed Apr. 2, 2007, now U.S. Pat. No. 7,955,848, whichclaims benefit under 35 USC 119(e) of provisional application60/788,067, filed Apr. 3, 2006.

This invention was made with government support under CA111422,CA087456, and CA130102 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention

All patent and non-patent references cited in the application, are alsohereby incorporated by reference, in their entirety.

BACKGROUND OF INVENTION MicroRNAs—Novel Regulators of Gene Expression

MicroRNAs (miRNAs) are an abundant class of short endogenous RNAs thatact as post-transcriptional regulators of gene expression bybase-pairing with their target mRNAs. The ˜22 nucleotide (nt) maturemiRNAs are processed sequentially from longer hairpin transcripts(primary miRNA/pri-miRNA or precursor miRNA) by the RNAse IIIribonucleases Drosha (Lee et al. 2003) and Dicer (Hutvagner et al. 2001,Ketting et al. 2001). To date more than 3400 miRNAs have been annotatedin vertebrates, invertebrates and plants according to the miRBasemicroRNA database re-lease 7.1 in October 2005 (Griffith-Jones 2004,Griffith-Jones et al. 2006), and many miRNAs that correspond to putativemiRNA genes have also been bioinformatically predicted. More than halfof all known mammalian miRNAs are hosted within the introns of pre-mRNAsor long ncRNA transcripts (Rodriquez et al. 2004). Many miRNA genes arearranged in genomic clusters (Lagos-Quintana et al. 2001). For example,ca. 40% of human miRNA genes appear in clusters of two or more, with thelargest cluster of 40 miRNA genes being located in the human imprinted14q32 domain (Setiz et al. 2004; Altuvia et al. 2005). In plants, 117miRNA genes have been identified in Arabidopsis thaliana while number ofmiRNAs identified in rice is currently 178 (Griffith-Jones 2004,Griffith-Jones et al. 2006). The identified miRNAs to date representmost likely the tip of the iceberg, and the number of miRNAs might turnout to be very large. Recent bioinformatic predictions combined witharray analyses, small RNA cloning and Northern blot validation indicatethat the total number of miRNAs in vertebrate genomes is significantlyhigher than previously estimated and may be as many as 1000 (Bentwich etal. 2005, Berezikov et al. 2005, Xie et al. 2005).

The first miRNAs genes to be discovered, lin-4 and let-7, base-pairincompletely to repeated elements in the 3′ untranslated regions (UTRs)of heterochronic genes, and control developmental timing in C. elegansby regulating translation directly and negatively via antisense RNA-RNAinteraction (Lee et al. 1993, Reinhart et al. 2000). The majority ofplant miRNAs have perfect or near-perfect complementarity with theirtarget sites and direct RISC-mediated target mRNA cleavage (for review,see Bartel 2004). A large fraction of the plant miRNAs appear toregulate genes with roles in developmental processes, such as control ofmeristem identity, cell proliferation, developmental timing andpatterning (Kidner and Martienssen 2005). In contrast, most animalmiRNAs recognise their target sites located in 3′-UTRs by incompletebase-pairing, resulting in translational repression of the target genes(Bartel 2004). An increasing body of research shows that animal miRNAsplay fundamental biological roles in cell growth and apoptosis(Brennecke et al. 2003), hematopoietic lineage differentiation (Chen etal. 2004), life-span regulation (Boehm and Slack 2005), photoreceptordifferentiation (Li and Carthew 2005), homeobox gene regulation (Yektaet al. 2004, Hornstein et al. 2005), neuronal asymmetry (Johnston et al.2004), insulin secretion (Poy et al. 2004), brain morphogenesis(Giraldez et al. 2005), muscle proliferation and differentiation (Chen,Mandel et al. 2005, Kwon et al. 2005, Sokol and Ambros 2005),cardiogenesis (Zhao et al. 2005) and late embryonic development invertebrates (Wienholds et al. 2005). Several studies have identifiedsub-classes of miRNAs directly implicated in the regulation of mammalianbrain development and neuronal differentiation (Krichevsky et al. 2003,Miska at al. 2004, Sempere et al. 2004, Smirnova et al. 2005).Interestingly, many neural miRNAs appear to be temporally regulated incortical cultures copurifying with polyribosomes, suggesting that theymay control localized translation of dendrite-specific mRNAs (Kim et al.2004).

The number of regulatory mRNA targets of vertebrate miRNAs has beenestimated by identifying conserved complementarity to the miRNA seedsequences (nucleotide 2-7 of the miRNA), suggesting that ˜30% of thehuman genes may be miRNA targets (Lewis et al. 2005). Computationalpredictions in Drosophila provide evidence that a given miRNA has onaverage ˜100 mRNA target sites in the fly, while another recent studyreported that vertebrate miRNAs may target ˜200 mRNAs each, furthersupporting the notion that miRNAs can regulate the expression of a largefraction of the protein-coding genes in multicellular eukaryotes(Brennecke et al. 2005, Krek et al. 2005). Most recent reports indicatethat miRNAs may not function as developmental switches, but rather playa role in maintaining tissue identity by conferring accuracy togene-expression programs (Giraldez et al. 2005, Lim et al. 2005, Starket al. 2005, Farh et al. 2005, Wienholds et al. 2005).

MicroRNAs in Human Disease

The expanding inventory of human miRNAs along with their highly diverseexpression patterns and high number of potential target mRNAs suggestthat miRNAs are involved in a wide variety of human diseases. One isspinal muscular atrophy (SMA), a paediatric neurodegenerative diseasecaused by reduced protein levels or loss-of-function mutations of thesurvival of motor neurons (SMN) gene (Paushkin et al. 2002). A mutationin the target site of miR-189 in the human SLITRK1 gene was recentlyshown to be associated with Tourette's syndrome (Abelson et al. 2005),while another recent study reported that the hepatitis C virus (HCV) RNAgenome interacts with a host-cell miRNA, the liver-specific miR-122a, tofacilitate its replication in the host (Jopling et al. 2005). Otherdiseases in which miRNAs or their processing machinery have beenimplicated, include fragile X mental retardation (FXMR) caused byabsence of the fragile X mental retardation protein (FMRP) (Nelson etal. 2003, Jin et al. 2004) and DiGeorge syndrome (Landthaler et al.2004). In addition, perturbed miRNA expression patterns have beenreported in many human cancers. For example, the human miRNA genesmiR-15a and miR-16-1 are deleted or down-regulated in the majority ofB-cell chronic lymphocytic leukemia (CLL) cases, where a uniquesignature of 13 miRNA genes was recently shown to associate withprognosis and progression (Calin et al. 2002, Calin et al. 2005). Therole of miRNAs in cancer is further supported by the fact that more than50% of the human miRNA genes are located in cancer-associated genomicregions or at fragile sites (Calin et al. 2004). Recently, systematicexpression analysis of a diversity of human cancers revealed a generaldown-regulation of miRNAs in tumours compared to normal tissues (Lu etal. 2005).

Interestingly, miRNA-based classification of poorly differentiatedtumours was successful, whereas mRNA profiles were highly inaccuratewhen applied to the same samples. miRNAs have also been shown to bederegulated in lung cancer (Johnson et al. 2005) and colon cancer(Michael et al. 2004), while the miR-1792 cluster, which is amplified inhuman B-cell lymphomas and miR-155 which is upregulated in Burkitt'slymphoma have been reported as the first human miRNA oncogenes (E is etal. 2005, He at al. 2005). Thus, human miRNAs may not only be highlyuseful as biomarkers for future cancer diagnostics, but are rapidlyemerging as attractive targets for disease intervention by antisenseoligonucleotide technologies.

Human Breast Cancer

Breast cancer is one of the most common cancers of women; it is acomplex, inadequately understood, and often a fatal disease. Studies inmany laboratories over the past few decades have demonstrated thatbreast cancer (and indeed cancer in general) results from a series ofmutations affecting multiple classes of genes. Natural selection favoursthe growth of cells containing mutations that confer growth advantagesand prevent the functioning of normal growth inhibitory mechanisms suchas apoptosis. Mutations affecting both proto-oncogenes and tumoursuppressor genes contribute to breast cancer and affect diverse cellularprocesses including signal transduction, DNA replication and repair,transcription, translation, apoptosis and differentiation. Factors knownto be important for characterization, diagnosis and prognosis of breasttumours include the status of the estrogen receptor (ER), epithelialgrowth factor receptor (EGFR), human EGF receptor 2 (HER2) and p53, andsome of these can be used as targets for therapeutic intervention(Colozza et al., 2005). From a clinical perspective, ER and HER2 areconsidered the main molecular targets, since effective drugs exist totreat these tumour types: tamoxifen and aromatase inhibitors for ER+tumours and Herceptin for HER2-overexpressing tumours, respectively.

Human Lung Cancer

Lung cancer is the leading cause of cancer deaths in both women and menin the United States and throughout the world. Lung cancer is the numberone cause of cancer deaths in men and has surpassed breast cancer as theleading cause of cancer deaths in women. In the United States in 2004,160,440 people were projected to die from lung cancer compared with aprojected 127,210 deaths from colorectal, breast, and prostate cancercombined. Only about 14% of all people who develop lung cancer survivefor 5 years.

The lungs are a common site for metastasis, i.e. spreading of tumours tonearby lymph nodes or to other organs via the blood system. Lung cancersare usually divided into 2 groups accounting for about 95% of all cases.The division is based on the type of cells that comprise the cancer. The2 types of lung cancer are classified based on the cell size of thetumour. They are called small cell lung cancer (SCLC) and non-small celllung cancer (NSCLC) where the latter includes several types of tumours.SCLC is less common, however they grow more rapid and are more likely tometastasize than NSCLCs. SCLCs have often already spread to other partsof the body when the disease is diagnosed thus it is of high importanceto detect lung cancer at an early stage using efficient markers.

About 5% of lung cancers are of rare cell types, such as carcinoidtumour, lymphoma, or metastatic (cancers from other parts of the bodythat spread to the lungs).

The specific types of primary lung cancers are Adenocarcinoma (an NSCLC)which is the most common type of lung cancer, making up 30-40% of allcases. A subtype of adenocarcinoma is called bronchoalveolar cellcarcinoma, which creates a pneumonia-like appearance on chest x-rayfilms. Squamous cell carcinoma (an NSCLC) is the second most common typeof lung cancer, making up about 30% of all lung cancers while large cellcancer makes up 10% and SCLC 20% of all cases and carcinoid lung canceraccounts for 1% of all cases.

Cancer Diagnosis and Identification of Tumour Origin

Cancer classification relies on the subjective interpretation of bothclinical and histo-pathological information by eye with the aim ofclassifying tumours in generally accepted categories based on the tissueof origin of the tumour. However, clinical information can be incompleteor misleading. In addition, there is a wide spectrum in cancermorphology and many tumours are atypical or lack morphologic featuresthat are useful for differential diagnosis. These difficulties mayresult in diagnostic confusion, with the need for mandatory secondopinions in all surgical pathology cases (Tomaszewski and LiVolsi 1999,Cancer 86: 2198-2200).

Another problem for cancer diagnostics is the identification of tumourorigin for metastatic carcinomas. For example, in the United States,51,000 patients (4% of all new cancer cases) present annually withmetastases arising from occult primary carcinomas of unknown origin (ACSCancer Facts & Figures 2001: American Cancer Society). Adenocarcinomasrepresent the most common metastatic tumours of unknown primary site.Although these patients often present at a late stage, the outcome canbe positively affected by accurate diagnoses followed by appropriatetherapeutic regimens specific to different types of adenocarcinoma(Hillen 2000, Postgrad. Med. J. 76: 690-693). The lack of uniquemicroscopic appearance of the different types of adenocarcinomaschallenges morphological diagnosis of adenocarcinomas of unknown origin.The application of tumour-specific serum markers in identifying cancertype could be feasible, but such markers are not available at present(Milovic et al. 2002, Med. Sci. Monit. 8: MT25-MT30). Microarrayexpression profiling has been used to successfully classify tumoursaccording to their site of origin (Ramaswamy et al. 2001, Proc. Natl.Acad. Sci. U.S.A. 98: 15149-15154), but the lack of a standard for arraydata collection and analysis make them difficult to use in a clinicalsetting. SAGE (serial analysis of gene expression), on the other hand,measures absolute expression levels through a tag counting approach,allowing data to be obtained and compared from different samples. Thedrawback of this method is, however, its low throughput, making itinappropriate for routine clinical applications. Quantitative real-timePCR is a reliable method for assessing gene expression levels fromrelatively small amounts of tissue (Bustin 2002, J. Mol. Endocrinol. 29:23-39). Although this approach has recently been successfully applied tothe molecular classification of breast tumours into prognostic subgroupsbased on the analysis of 2,400 genes (Iwao et al. 2002, Hum. Mol. Genet.11: 199-206), the measurement of such a large number of randomlyselected genes by PCR is still not clinically practical.

Another limitation to further study and identify potential biomarkers isthe difficulty of conducting retrospective studies with archived tumoursamples (Ludwig and Weinstein, 2005). To be useful for subsequentstudies, tumour samples obtained from the operating room must betransported to the pathology laboratory in a timely manner, where thesamples are sectioned and stored as frozen or formalin-fixed andparaffin-embedded (FFPE) for archiving in a tumour bank. The quality ofthe RNA of frozen and FFPE samples is often compromised and is, thus,unsuitable for conducting accurate molecular tests. Hence, the smallsize of miRNAs offers a unique advantage due to the fact that theseshort RNA molecules are more stable and less prone to enzymaticdegradation by RNAses, and are therefore amenable to an accurateassessment of miRNA levels in archival tumour samples.

SUMMARY OF INVENTION

The present invention solves the above described problems by providingnovel miRNA sequences useful as biomarkers for diagnostic purposes ofhuman breast and lung cancers.

The present invention furthermore provides novel miRNA sequences whichare useful as forming the basis for molecular targeting for cancerintervention, in which for example LNA-modified, stabilized syntheticmiRNA species (miRNA agonist) are used to replace the activity of amissing or a downregulated miRNA or an LNA-modified antagonistic-miRoligonucleotide used to inhibit the activity of an amplified orover-expressed miRNA, as well as for engineered over-expression of arepressed species in desired pulmonary epithelial cells that basally donot express them.

The invention furthermore provides novel miRNA sequences, which allowidentification of the target genes of these miRNAs, thereby enabling usto understand how the altered expression of these miRNAs affectscellular properties important for oncogenesis (including activation orrepression in dysplastic tissues) and for the maintenance or progressionof early pre-malignant lesions. This, in turn, can be used to identifyadditional molecular targets for development of novel, targeted breastand lung cancer treatment or chemoprevention.

The invention furthermore provides protocols for clinical application ofmiRNA detection as diagnostic and/or prognostic tools which would permitdetecting these diagnostic miRNAs with less invasive techniques thanbiopsy, such as in ductal fluid samples (from nipple aspirates or ductallavage) or in peripheral blood, in order to screen for early stages ofbreast cancer. This allows predicting outcomes of pre-treatment and topredict which therapeutic agents would be most effective. The inventionfurthermore provides novel miRNA sequences and methods based on thesesequences allowing monitoring of changes in the miRNA expressionpatterns useful as early indicators of response to treatment.

These provisions are encompassed by the embodiments of the presentinvention characterised in the claims.

It is thus an object of the present invention to provide a method fordetection, classification, diagnosis and prognosis of a diseasecomprising the steps of obtaining at least one sample from an individualand detecting the presence or absence of expression and/or theexpression level of at least one nucleic acid molecule described herein.

It is likewise an object of the present invention to provide a methodfor detection, classification, diagnosis and prognosis of breast or lungcancer comprising the steps of obtaining at least one sample from anindividual and detecting the presence or absence of expression and/orthe expression level of at least one nucleic acid molecule describedherein.

Thus it is an aspect of the present invention to provide a probe for thedetection of said nucleic acid molecule.

It is furthermore an aspect of the present invention to provide anucleic acid molecule as specified herein for the production of apharmaceutical composition, and a pharmaceutical composition comprisingat least one nucleic acid molecule as described herein.

An aspect of the invention regards a kit comprising at least one probefor detection of at least one nucleic acid molecule as described herein.

It is also an aspect of the present invention to provide a nucleic acidconstruct encoding at least one nucleic acid molecule described herein.

It is a further aspect of the present invention to provide a deliveryvehicle comprising the nucleic acid molecule described herein or thenucleic acid construct of the above.

Yet an aspect of the present invention provides a cell comprising atleast one nucleic acid molecule described herein.

It follows that the present invention also provides a pharmaceuticalcomposition comprising any of the nucleic acid constructs, cells ordelivery vehicles mentioned herein.

An object of the present invention regards a method of modulating theexpression of a pre-miRNA or miRNA in a cell, tissue or animalcomprising contacting the cell, tissue or animal with any of thecompositions of the present invention.

Yet an object of the present invention regards a method of treating orpreventing a disease or disorder associated with aberrant expression ofa pre-miRNA, miRNA or the target of a miRNA in a cell, tissue or animalcomprising contacting the cell, tissue or animal with any of thecompositions of the present invention.

The present invention also provides for the use of a nucleic acidmolecule described herein, for the production of a pharmaceuticalcomposition for the diagnosis or treatment of cancer.

DESCRIPTION OF DRAWINGS

FIG. 1: Northern blots used for quantification of data presented intable IV (upper panel) and table V (lower panel). Numbers directly abovepanels refer to BC # numbers in tables IV and V.

FIG. 2: Detection of miRNA expression by in situ hybridization usingFITC-labeled LNA probes in FFPE sections from breast cancer patients.

FIG. 3: Detection of miRNA expression by in situ hybridization usingFITC-labeled LNA probes in FFPE sections from a breast cancer patientcase showing a progression series from normal to malignant tissue.miR-145 expression is detected in fewer cells and at lower levels as thecells become malignant.

FIG. 4: Detection of miRNA expression by in situ hybridization usingFITC-labeled LNA probes in 0.6 mm cores assembled in a tissue microarrayfrom FFPE blocks of 59 breast cancer patients. Images of representativecases, used for data analysis in table VII, are shown.

FIG. 5: Cluster analysis of miRNA expression in mouse lung specimens.

FIG. 6: Semi-quantitative RT-PCR assays for a representative miRNA(mi34c) differentially repressed in malignant versus normal lung from(A) transgenic mouse lines and from (B) paired human normal-malignantlung tissues.

FIG. 7: Detection of miRNA expression by in situ hybridization usingFITC-labeled LNA probes in FFPE sections of murine model of lung cancer.

FIG. 8: Over-expressed miRNAs in murine transgenic lung cancers relativeto adjacent normal lung tissues.

FIG. 9: ISH assays for representative over-expressed miRNAs in lungcancers

FIG. 10: Validation of miR-136, miR-376a and miR-31 expression profilesby real-time RT-PCR assays.

FIG. 11: Validation of miR-31 expression by real-time RT-PCR assays.

FIG. 12: Regulation of miR-31 expression affects lung cancer cellproliferation.

FIG. 13: Repression of miR-31 expression significantly affects murinelung cancer clonal growth and tumorigenicity.

FIG. 14: LATS2 and PPP2R2A are miR-31 target mRNAs.

FIG. 15: Proliferation of murine lung cancer cells following regulatedexpression of miR-31, LATS2 or PPP2R2A.

FIG. 16: Validation of LATS2 and PPP2R2A expression profiles byreal-time RT-PCR assays

DETAILED DESCRIPTION OF THE INVENTION Definitions

Chemotherapeutic: A drug used to treat a disease, especially cancer. Inrelation to cancer the drugs typically target rapidly dividing cells,such as cancer cells.Complementary or substantially complementary: Refers to thehybridization or base pairing between nucleotides or nucleic acids, suchas, for instance, between the two strands of a double stranded DNAmolecule or between an oligonucleotide primer and a primer binding siteon a single stranded nucleic acid to be sequenced or amplified.Complementary nucleotides are, generally, A and T (or A and U), or C andG. Two single stranded RNA or DNA molecules are said to be substantiallycomplementary when the nucleotides of one strand, optimally aligned andwith appropriate nucleotide insertions or deletions, pair with at leastabout 80% of the nucleotides of the other strand, usually at least about90% to 95%, and more preferably from about 98 to 100%.Complementary DNA (cDNA): Any DNA obtained by means of reversetranscriptase acting on RNA as a substrate. Complementary DNA is alsotermed copy DNA.Complementary strand: Double stranded polynucleotide contains twostrands that are complementary in sequence and capable of hybridizing toone another.Cytostatic: A drug that inhibits or suppresses cellular growth andmultiplication.Delivery vehicle: An entity whereby a nucleotide sequence or polypeptideor both can be transported from at least one media to another.Fragment: is used to indicate a non-full length part of a nucleic acidor polypeptide. Thus, a fragment is itself also a nucleic acid orpolypeptide, respectively.In situ detection: The detection of expression or expression levels inthe original site hereby meaning in a tissue sample such as biopsy.LNA: Locked nucleic acid. LNA is a modified RNA nucleotide in which thesugar ring is conformationally locked in the 3′ endo conformation byintroduction of a O2′,C4′-methylene linkage thereby increasing itsthermal stability.miRNA: miRNA or microRNA refer to 19-25 nt non-coding RNAs derived fromendogenous genes that act as post-transcriptional regulators of geneexpression. They are processed from longer (ca 70-80 nt) hairpin-likeprecursors termed pre-miRNAs by the RNAse III enzyme Dicer. MiRNAsassemble in ribonucleoprotein complexes termed miRNPs and recognisetheir target sites by antisense complementarity thereby mediatingdown-regulation of their target genes. Near-perfect or perfectcomplementarity between the miRNA and its target site results in targetmRNA cleavage, whereas limited complementarity between the miRNA and thetarget site results in translational inhibition of the target gene.miRNA targeting moiety: Is any moiety or entity capable of binding,inhibiting, or interfering with the activity and/or expression of amiRNA, this being either a (such as one or more) mature or precursormiRNA. Preferably, the moiety is capable of binding the target miRNA.Nucleic acid: The term “nucleic acid” refers to polydeoxyribonucleotides(containing 2-deoxy-D-ribose), to polyribonucleotides (containingD-ribose), and to any other type of polynucleotide which is an Nglycoside of a purine or pyrimidine base, or modified purine orpyrimidine bases. The term refers only to the primary structure of themolecule. Thus, the term “nucleic acid” includes double- andsingle-stranded DNA of different lengths, as well as double- and singlestranded RNA of different lengths, or synthetic variants hereof such asfor example LNA, of different lengths. Thus, the nucleic acid may forexample be DNA, RNA, LNA, HNA, PNA, or any other variant hereof.Chemically modified oligonucleotides are especially relevant aspects ofthe present invention; these are known to the skilled person. As usedherein the term nucleotide includes linear oligomers of natural ormodified monomers or linkages, including deoxyribonucleotides,ribonucleotides, anomeric forms thereof, peptide nucleic acid monomers(PNAs), locked nucleotide acid monomers (LNA), and the like, capable ofspecifically binding to a single stranded polynucleotide tag by way of aregular pattern of monomer-to-monomer interactions, such as Watson-Cricktype of base pairing, base stacking, Hoogsteen or reverse Hoogsteentypes of base pairing, or the like. Usually monomers are linked byphosphodiester bonds or analogs thereof to form oligonucleotides rangingin size from a few monomeric units, e.g. 3-4, to several tens ofmonomeric units, e.g. 40-60. Whenever an oligonucleotide is representedby a sequence of letters, such as “ATGCCTG,” it will be understood thatthe nucleotides are in 5′ 3′ order from left to right and the “A”denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotesdeoxyguanosine, and “T” denotes thymidine, unless otherwise noted. Inaddition to the 100% complementary form of double-strandedoligonucleotides, the term “double-stranded” as used herein is alsomeant to refer to those forms which include such structural features asbulges and loops.Operative linker: A nucleic acid sequence or a peptide that bindtogether two sequences in a nucleic acid construct or (chimeric)polypeptide in a manner securing the biological processing of thenucleic acid or polypeptide.Plurality: At least two.pri-miRNA: Refers to the primary miRNA transcript. Initially, miRNAgenes are transcribed by RNA polymerase II into long primary miRNAs(pri-miRNAs). The processing of these pri-miRNAs into the final maturemiRNAs occurs stepwise and compartmentalized. In animals, pri-miRNAs areprocessed in the nucleus into 70-80-nucleotide precursor miRNAs(pre-miRNAs) by the RNase III enzyme Drosha. Both pri-miRNA and pre-RNAare objects of the present invention and the terms may be usedinterchangeably herein.Probe: The term “probe” refers to a defined oligonucleotide sequence ora nucleic acid sequence which said sequence is used to detect target DNAor RNA nucleic acid sequences by hybridization, bearing a complementarysequence to the probe. A probe may be labelled to facilitate detection.Promoter: Refers to a regulatory region of DNA a short distance upstreamfrom the 5′ end of a transcription start site that acts as the bindingsite for RNA polymerase. A region of DNA to which RNA polymerase bindsin order to initiate transcription.siRNA: (Small interfering RNAs) The term “siRNA” refers to 21-25 nt RNAsderived from processing of linear double-stranded RNA. siRNAs assemblein complexes termed RISC(RNA-induced silencing complex) and targethomologous RNA sequences for endonucleolytic cleavage. Synthetic siRNAsalso recruit RISCs and are capable of cleaving homologous RNA sequences.Surfactant: A surface active agent capable of reducing the surfacetension of a liquid in which it is dissolved. A surfactant is a compoundcontaining a polar group which is hydrophilic and a non polar groupwhich is hydrophobic and often composed of a fatty acid chain.Vaccine: A substance or composition capable of inducing a protectiveimmune response in an animal. A protective immune response being animmune response (humoral/antibody and/or cellular) inducing memory in anorganism, resulting in the infectious agent, being met by a secondaryrather than a primary response, thus reducing its impact on the hostorganism.Variant: a ‘variant’ of a given reference nucleic acid or polypeptiderefers to a nucleic acid or polypeptide that displays a certain degreeof sequence homology to said reference nucleic acid or polypeptide butis not identical to said reference nucleic acid or polypeptide.Vector: A genetically engineered nucleic acid also referred to asnucleic acid construct: Typically comprising several elements such asgenes or fragments of same, promoters, enhancers, terminators, polyAtails, linkers, selection markers or others. A vector may enableexpression of cloned genes or gene fragments in a particular cell type,or be used to transfer genetic information by insertion etc.Vehicle: An agent with which genetic material can be transferred e.g. amodified virus or a chemical or other transfection agent.

The present invention relates to methods of detecting, classifying,diagnosing and providing a prognosis for hyperproliferative diseasessuch as cancer and especially breast and lung cancer by the detection ofnucleic acid molecules. The invention furthermore relates topharmaceutical compositions comprising nucleic acid molecules and theiruse in treatment of said diseases.

miRNA

An aspect of the present invention relates to the use and detection ofmicroRNA (miRNA) sequences especially regarding cancer and specificallyhuman breast and lung cancer respectively.

By miRNA is understood a single-stranded RNA of typically 19-25nucleotides length, also referred to as a mature miRNA. Suchsingle-stranded RNAs may be isolated from an organism, but may also besynthesized by standard techniques. miRNA is in an organism synthesizedas pri-miRNA or primary miRNA transcript which is enzymatically cleavedinto pre-miRNA and finally after a second round of enzymatic cleavageresults in miRNA.

The present invention relates to the discovery of differentialexpression levels of various miRNAs in cancerous tissue compared tonormal tissue, see examples for specifics. When compared to biopsiesfrom corresponding healthy tissue both by analysing whole samples byNorthern and LNA microarray techniques and by analysing spatialdistribution of miRNAs within epithelial structures of breast tissue byin situ hybridization techniques, the following miRNAs are downregulatedin breast tumours: hsa-miR-451, hsa-miR-143 and hsa-miR-145. Incontrast, the following miRNAs are upregulated in specific subtypes ofbreast tumours compared to normal breast biopsies: hsa-miR-141,hsa-miR-200b, hsa-miR-200c, hsa-miR-221, hsa-miR-222 and hsa-miR-21. Theresults of the experiments are summarised in Table A, see Example 3 formore details. The hereby disclosed discovery is obviously of greatinterest and consequently it is worth noting that all of these miRNAsare aspects of the present invention. The downregulation of hsa-miR-451and the upregulation of hsa-miR-200b, hsa-miR-200c, hsa-miR-221 andhsa-miR-222 are of significant interest, especially in relation tobreast cancer, and especially the discovery of the downregulation ofhsa-miR-451 is of great importance.

The present invention furthermore relates to the discovery ofdifferential expression levels of various miRNAs in cancerous lungtissue compared to normal tissue, see examples for specifics. Whencompared to biopsies from corresponding healthy tissue both by analysingwhole samples by Northern and LNA microarray techniques and by analysingspatial distribution of miRNAs within epithelial structures of lungtissue by in situ hybridization techniques, the following miRNAs aredown-regulated in lung tumours: hsa-miR-34b, hsa-miR-34c,hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-486, hsa-miR-451, hsa-miR-145,hsa-miR-144 and hsa-miR-150. In contrast, the following miRNAs areupregulated in lung tumours compared to biopsies from healthy lungtissue: hsa-miR-31, hsa-miR-127, hsa-miR-141, hsa-miR-136 andhsa-miR-376a.

The results of the experiments are summarised in Table A, see examplesfor more details. The hereby disclosed discovery is obviously of greatinterest and consequently it is worth noting that all of these miRNAsare aspects of the present invention. The downregulation of hsa-miR-34cand hsa-miR150 are of significant interest, especially in relation tolung cancer.

TABLE A SEQ ID NO Name Form Tumour vs normal expr. 1 hsa-miR-451 maturedown 2 hsa-miR-143 mature down 3 hsa-miR-145 mature down 4 hsa-miR-141mature up 5 hsa-miR-200b mature up 6 hsa-miR-200c mature up 7hsa-miR-221 mature up 8 hsa-miR-222 mature up 9 hsa-miR-451 precursor Nodata (ND) 10 hsa-miR-143 precursor (ND) 11 hsa-miR-145 precursor (ND) 12hsa-miR-141 precursor (ND) 13 hsa-miR-200b precursor (ND) 14hsa-miR-200c precursor (ND) 15 hsa-miR-221 precursor (ND) 16 hsa-miR-222precursor (ND) 17 hsa-miR-21 mature up 18 hsa-miR-21 precursor (ND) 19hsa-miR-34b mature down 20 hsa-miR-34c mature down 21 hsa-miR-142-3pmature down 22 hsa-miR-142-5p mature down 23 hsa-miR-486 mature down 24hsa-miR-144 mature down 25 hsa-miR-31 mature up 26 hsa-miR-127 mature up27 hsa-miR-136 mature up 28 hsa-miR-376a mature up 29 hsa-miR-150 maturedown

All of the miRNAs of Table A are thus differentially expressed inhyperproliferative tissue compared to normal tissue and all are ofspecial interest with regard to the present invention. Both miRNAs, theexpression of which is upregulated in hyperproliferative tissue areaspects of the present invention as are miRNAs the expression of whichis downregulated in hyperproliferative tissues compared to normaltissue. A preferred embodiment of the present invention regards all themiRNAs or nucleic acid molecules as defined herein below in respect tohyperproliferative tissue such as cancerous tissue. A more preferredembodiment regards the miRNAs/the nucleic acid molecules as definedherein of SEQ ID NOs 1 to 18 of Table A in regards to breast cancertissue and SEQ ID NOs: 1, 3, 4 and 19 to 29 of Table A in regards tolung cancer tissue. Thus SEQ ID NOs: 1, 3 and 4 are an embodiment ofboth present invention regarding both lung and breast cancer. An evenmore preferred embodiment regards the miRNAs/nucleic acid molecules ofSEQ ID NO: 1, 5, 6, 7, 8, 9, 13, 14, 15 and 16 in regards to breastcancer and SEQ ID NO: 1, 3, 4 and 19 to 29 in regards to lung cancer. Amost preferred embodiment regards the miRNA of SEQ ID NO: 1 and 9 inregards to breast cancer and SEQ ID NO: 19 and 29 in regards to lungcancer.

The present invention thus regards the sequences identified herein asSEQ ID NOs: 1 to 29. These sequences are exact sequences of miRNAs andtheir precursors as found in the human body. The sequences of thepresent invention may be isolated here from or may be synthesized bystandard techniques known in the art.

Bases not commonly found in natural nucleic acids may be included in thenucleic acids of the present invention and these include, for example,inosine and 7-deazaguanine. Furthermore, synthetic nucleotides thatconfer an added stability to the nucleotides of the present invention,such as LNA, fall within the scope of the present invention. LNA is asynthetic RNA analog that increases the thermal stability ofoligonucleotides. Other modifications of the nucleotides of the presentinvention that confer added metabolic or thermal stability to these arealso included within the scope of the present invention.

The invention also regards sequences which are complementary to any ofsequences SEQ ID NO: 1 to 29. The complementary sequence of a nucleicacid sequence as used herein refers to an oligonucleotide or a nucleicacid sequence that can form a double-stranded structure by means ofWatson-Crick base pairing, i.e. by formation of hydrogen bonds betweenthe complementary nucleobases. Thus complementary refers to the capacityfor precise pairing between two nucleic acid sequences with one another.For example, if a nucleotide at a certain position of an oligonucleotideis capable of hydrogen bonding with a nucleotide at the correspondingposition of a DNA, RNA or other nucleic acid molecule as defined herein,then the oligonucleotide and the DNA or RNA are considered to becomplementary to each other at that position. The DNA or RNA strand areconsidered complementary to each other when a sufficient number ofnucleotides in the oligonucleotide can form hydrogen bonds withcorresponding nucleotides in the target DNA or RNA to enable theformation of a stable complex. Thus, complementarity may not be perfect;stable duplexes may contain mismatched base pairs or unmatched bases.Those skilled in the art of nucleic acid technology can determine duplexstability empirically considering a number of variables including, forexample, the length of the oligonucleotide, percent concentration ofcytosine and guanine bases in the oligonucleotide, ionic strength, andincidence of mismatched base pairs.

The invention also relates to fragments of said sequences. For themature miRNAs (SEQ ID NOs: 1 to 8, 17 and 19 to 29) said fragments areat least 10 nucleotides in length, such as 11, 12, 13, 14, 15, 16, 17 or18 nucleotides in length. Preferably, the fragments are 19, 20, 21, 22,or 23 nucleotides in length. For the precursor miRNA sequences, thefragments comprise either the sequence of the mature miRNA or itscomplementary sequence or both. The precursor miRNA sequences are atleast 40 nucleotides in length, such as at least 50, 60, 65, 70, 75, 80,85, 90, 95, 100 or 105 nucleotides in length.

In one preferred embodiment of the invention there is also providedvariants of the sequences SEQ ID NO: 1 to 8 and 17 and variants offragments thereof. In these variants one or more nucleotides have beensubstituted for (an)other nucleotide(s) or nucleotides may be added ordeleted from the variant compared to the predetermined sequence. Thevariant sequences may herein be referred to as such or functionalequivalents or substituted sequences; these expressions are usedinterchangeably herein.

Variants are characterized by their degree of identity to thepredetermined sequence identified by a given SEQ ID NO from which thevariant sequence is derived or to which it can be said to be related.The degree of identity shared between any of the sequences identified asSEQ ID NO 1 to 29 and a variant sequence is preferably at least 55%sequence identity, such as 60% sequence identity, for example 65%, suchas at least 70% sequence identity or 75% sequence identity. Morepreferably the variant sequence shares at least 80% sequence identity toany of sequences SEQ ID NO: 1 to 29, such as 85% sequence identity, forexample 90% sequence identity, such as at least 92% sequence identity or94% sequence identity. Most preferably the variant sequences share atleast 95%, for example 96% sequence identity, such as 97% sequenceidentity, such as at least 98% sequence identity, or most preferably thevariant shares 99% sequence identity with the predetermined sequence.

The term “identity” or “sequence identity” means that two polynucleotidesequences are identical (i.e., on a nucleotide-by-nucleotide basis) overthe window of comparison. The term “percentage of sequence identity” iscalculated by comparing two optimally aligned sequences over the windowof comparison, determining the number of positions at which theidentical nucleotide (e.g., A, T, C, G, U, or I) occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison (i.e., the window size), and multiplying the result by 100 toyield the percentage of sequence identity. The identity of the sequencesis calculated by standard sequence identity calculation methods.

The present invention also regards nucleic acid molecules which understringent conditions can hybridize to any of the nucleic acid moleculesdescribed in the above. Stringent conditions as used herein denotestringency as normally applied in connection with Southern blotting andhybridization as described e.g. by Southern E. M., 1975, J. Mol. Biol.98:503-517. For such purposes it is routine practice to include steps ofprehybridization and hybridization. Such steps are normally performedusing solutions containing 6×SSPE, 5% Denhardt's, 0.5% SDS, 50%formamide, 100 μg/ml denatured salmon sperm DNA (incubation for 18 hrsat 42° C.), followed by washings with 2×SSC and 0.5% SDS (at roomtemperature and at 37° C.), and a washing with 0.1×SSC and 0.5% SDS(incubation at 68° C. for 30 min), as described by Sambrook et al.,1989, in “Molecular Cloning/A Laboratory Manual”, Cold Spring Harbor),which is incorporated herein by reference.

When referring to a nucleotide sequence or nucleic acid molecule in theremainder of the text, it shall be understood to be a nucleotidesequence or nucleic acid molecule comprising:

-   -   a) a nucleotide sequence selected from the group consisting of        SEQ ID NO 1 to 29.    -   b) a nucleotide sequence which is complementary to a) and/or,    -   c) a nucleotide sequence which is a fragment of a) or b) and/or,    -   d) a nucleotide sequence which has an identity of at least 80%        to a sequence of a), b) or c) and/or,    -   e) a nucleotide sequence which hybridizes under stringent        conditions to a sequence of a), b), c) or d).

Cancer

An aspect of the present invention regards the detection,classification, prevention, diagnosis, prognosis and treatment ofcancer, especially breast and lung cancers.

As used herein, the terms “cancer,” “hyperproliferative,” and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state or condition characterized by rapidlyproliferating cell growth. Hyperproliferative and neoplastic diseasestates may be categorized as pathologic, i.e., characterizing orconstituting a disease state, or may be categorized as nonpathologic,i.e., a deviation from normal but not associated with a disease state.The term is meant to include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathologic type or stage ofinvasiveness. “Pathologic hyperproliferative” cells occur in diseasestates characterized by malignant tumour growth. Examples ofnonpathologic hyperproliferative cells include proliferation of cellsassociated with wound repair.

The terms “cancer” or “neoplasms” include malignancies of the variousorgan systems, such as affecting lung, breast, thyroid, lymphoid,gastrointestinal, and genito-urinary tract, as well as adenocarcinomaswhich include malignancies such as most colon cancers, renalcellcarcinoma, prostate cancer and/or testicular tumours, non-small cellcarcinoma of the lung, cancer of the small intestine and cancer of theesophagus.

Preferably, the present invention regards breast and lung cancerrespectively. Breast cancer is a cancer of the breast tissue. There aremany different types of breast cancer, but the vast majority, over 80%,begins in either the milk ducts or the lobular tissue. Breast cancerscan be classified histologically based upon the types and patterns ofcells that compose them. Carcinomas can be invasive; extending into thesurrounding stroma, or non-invasive; confined just to the ducts orlobules. Invasive carcinomas of the breast include: Infiltrating DuctalCarcinoma, Infiltrating Lobular Carcinoma, Infiltrating Ductal & LobularCarcinoma, Medullary Carcinoma, Mucinous (colloid) Carcinoma,Comedocarcinoma, Paget's disease, Papillary Carcinoma, TubularCarcinoma, Adenocarcinoma and Carcinoma. Non-invasive carcinomas of thebreast include: Intraductal Carcinoma, Lobular Carcinoma in situ (LCIS),Intraductal & LCIS, Papillary Carcinoma and Comedocarcinoma.

Microarray expression profiling of mRNAs in breast cancers indicate thatthey can be grouped into five distinct subtypes that differ with respectto their patterns of gene expression, detailed phenotypes, prognosis,and susceptibility to specific treatments (Perou et al., 1999, Sortie etal., 2001, 2003, 2004, 't Veer et al., 2002). These five subtypes are:luminal A, luminal B, HER2-overexpressing, basal and normal-like. Thesubtypes exhibit distinct gene signatures that correlate with clinicaloutcome ('t Veer et al., 2002, Sortie et al., 2003). For example,luminal A subtype is mainly ER+ and its gene signature includesexpression of ER, FBP1, GATA3 and other genes. This subtype has the mostfavourable prognosis and clinical outcome. Similarly,HER2-overexpresssing subtype comprises mainly HER2-amplified tumoursthat express high levels of e.g. HER2, FLOT1, GRB7. This subtype has aless favourable prognosis and clinical outcome.

It is an object of the present invention to provide a method to detect,classify, diagnose and enable a prognosis of cancer. Preferably it is anobject of the present invention to provide a method to detect, classify,diagnose and enable a prognosis of breast cancer. The breast cancer maybe any breast cancer, specifically any breast cancer selected from thegroup of: luminal A, luminal B, HER2-overexpressing, basal andnormal-like subtypes of breast cancer.

A preferred embodiment of the present invention provides a method todetect, classify, diagnose and enable a prognosis of a breast cancercomprising at least one nucleic acid molecule such as SEQ ID NO: 1, 5,6, 7, 8, 9, 13, 14, 15 and 16.

A more preferred embodiment of the present invention provides a methodto detect, classify, diagnose and enable a prognosis of ahyperproliferative disease comprising detecting a nucleic acid moleculesuch as SEQ ID NO: 1 or 9.

A most preferred embodiment of the present invention provides a methodto detect, classify, diagnose and enable a prognosis of a cancercomprising any of the above molecules in combination with at least onenucleic acid molecule such as SEQ ID NO: 1 to 29.

Another highly preferred embodiment of the present invention regardslung cancer.

Lung cancer is the malignant transformation and expansion of lungtissue, and is the most lethal of all cancers worldwide, responsible for1.2 million deaths annually. It is caused predominantly by cigarettesmoking, and predominantly affected men, but with increased smokingamong women, it is now the leading cause of death due to cancer inwomen. However, some people who have never smoked still get lung cancer.

It is an object of the present invention to provide a method to detect,classify, diagnose and enable a prognosis of cancer. Preferably it is anobject of the present invention to provide a method to detect, classify,diagnose and enable a prognosis of lung cancer. The lung cancer may beany lung cancer, specifically any lung cancer selected from the group ofsmall cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC).

A preferred embodiment of the present invention provides a method todetect, classify, diagnose and enable a prognosis of a lung cancercomprising detecting at least one nucleic acid molecule such as SEQ IDNO: 19 to 29.

A more preferred embodiment of the present invention provides a methodto detect, classify, diagnose and enable a prognosis of ahyperproliferative disease comprising detecting a nucleic acid moleculesuch as SEQ ID NO: 19 or 29.

A most preferred embodiment of the present invention provides a methodto detect, classify, diagnose and enable a prognosis of a cancercomprising any of the above molecules in combination with at least onenucleic acid molecule such as SEQ ID NO: 1 to 29.

Sample

The present invention provides a method of detection, classification,diagnosis or prognosis of hyperproliferative diseases on at least onesample obtained from an individual. The individual may be any mammal,but is preferably a human. The individual may be any individual, anindividual predisposed of a disease or an individual suffering from adisease, wherein the disease is a hyperproliferative disease.

A sample as defined herein is a small part of an individual,representative of the whole and may be constituted by a biopsy or a bodyfluid sample. Biopsies are small pieces of tissue and may be fresh,frozen or fixed, such as formalin-fixed and paraffin embedded (FFPE).Body fluid samples may be blood, plasma, serum, urine, sputum,cerebrospinal fluid, milk, or ductal fluid samples and may likewise befresh, frozen or fixed. Samples may be removed surgically, by extractioni.e. by hypodermic or other types of needles, by microdissection orlaser capture.

As the object of the present invention regards hyperproliferativediseases especially cancers and specifically breast and lung cancers,obtaining more than one sample, such as two samples, such as threesamples, four samples or more from individuals, and preferably the sameindividual, is of importance. The at least two samples may be taken fromnormal tissue and hyperproliferative tissue, respectively. This allowsthe relative comparison of expression both as in the presence or absenceof at least one nucleic acid and/or the level of expression of the atleast one nucleic acid between the two samples. Alternatively, a singlesample may be compared against a “standardized” sample, such a samplebeing a sample comprising material or data from several samples,preferably also from several individuals. A standardized sample maycomprise either normal or hyperproliferative sample material or data.

In a preferred embodiment of the present invention the method ofdetection, classification, diagnosis or prognosis is performed on abiopsy. In a more preferred embodiment of the present invention themethod of detection, classification, diagnosis or prognosis is performedon a body fluid sample such as a blood sample or a ductal fluid sample.Ductal fluid samples may be from nipple aspirates or ductal lavage.

Sample Preparation

Before analyzing the sample, it will often be desirable to perform oneor more sample preparation operations upon the sample. Typically, thesesample preparation operations will include such manipulations asconcentration, suspension, extraction of intracellular material, e.g.,nucleic acids from tissue/whole cell samples and the like, amplificationof nucleic acids, fragmentation, transcription, labelling and/orextension reactions.

Nucleic acids, especially RNA and specifically miRNA can be isolatedusing any techniques known in the art. There are two main methods forisolating RNA: phenol-based extraction and silica matrix or glass fiberfilter (GFF)-based binding. Phenol-based reagents contain a combinationof denaturants and RNase inhibitors for cell and tissue disruption andsubsequent separation of RNA from contaminants. Phenol-based isolationprocedures can recover RNA species in the 10-200-nucleotide range e.g.,miRNAs, 5S rRNA, 5.8S rRNA, and U1 snRNA. If a sample of “total” RNA waspurified by the popular silica matrix column or GFF procedure, it may bedepleted in small RNAs. Extraction procedures such as those using Trizolor TriReagent, however will purify all RNAs, large and small, and arethe recommended methods for isolating total RNA from biological samplesthat will contain miRNAs/siRNAs.

Any method required for the processing of a sample prior to detection byany of the herein mentioned methods falls within the scope of thepresent invention. These methods are typically well known by a personskilled in the art.

Detection

It is within the general scope of the present invention to providemethods for the detection of miRNA. An aspect of the present inventionrelates to the detection of the miRNA sequences of table A or of anynucleic acid molecule as defined in the above. By detection is meantboth 1) detection in the sense of presence versus absence of one or moremiRNAs as well as 2) the registration of the level or degree ofexpression of one or more miRNAs, depending on the method of detectionemployed.

The detection of one or more nucleic acid molecules allows for theclassification, diagnosis and prognosis of a disease such as ahyperproliferative disease, e.g. a cancer and specifically a breast orlung cancer. The classification of a disease is of relevance bothmedically and scientifically and may provide important informationuseful for the diagnosis, prognosis and treatment of the disease. Thediagnosis of a disease is the affirmation of the presence of the diseasebased, as is the object of the present invention, on the expression ofat least one miRNA or miRNA precursor molecule herein also referred toas a nucleic acid molecule. Prognosis is the estimate or prediction ofthe probable outcome of a disease and the prognosis of a disease isgreatly facilitated by increasing the amount of information on theparticular disease. The method of detection is thus a central aspect ofthe present invention.

Any method of detection falls within the general scope of the presentinvention. The detection methods may be generic for the detection ofnucleic acids especially RNA, or be optimized for the detection of smallRNA species, as both mature and precursor miRNAs fall into this categoryor be specially designed for the detection of miRNA species. Thedetection methods may be directed towards the scoring of a presence orabsence of one or more nucleic acid molecules or may be useful in thedetection of expression levels.

The detection methods can be divided into two categories herein referredto as in situ methods or screening methods. The term in situ methodrefers to the detection of nucleic acid molecules in a sample whereinthe structure of the sample has been preserved. This may thus be abiopsy wherein the structure of the tissue is preserved. In situ methodsare generally histological i.e. microscopic in nature and include butare not limited to methods such as: in situ hybridization techniques andin situ PCR methods

Screening methods generally employ techniques of molecular biology andmost often require the preparation of the sample material in order toaccess the nucleic acid molecules to be detected. Screening methodsinclude, but are not limited to methods such as: Array systems, affinitymatrices, Northern blotting and PCR techniques, such as real-timequantitative RT-PCR.

Probe

It is an object of the present invention to provide a probe which can beused for the detection of a nucleic acid molecule as defined herein. Aprobe as defined herein is a specific sequence of a nucleic acid used todetect nucleic acids by hybridization. A nucleic acid is also here anynucleic acid, natural or synthetic such as DNA, RNA, LNA or PNA. A probemay be labelled, tagged or immobilized or otherwise modified accordingto the requirements of the detection method chosen. A label or a tag isan entity making it possible to identify a compound to which it isassociated. It is within the scope of the present invention to employprobes that are labelled or tagged by any means known in the art such asbut not limited to: radioactive labelling, fluorescent labelling andenzymatic labelling. Furthermore the probe, labelled or not, may beimmobilized to facilitate detection according to the detection method ofchoice and this may be accomplished according to the preferred method ofthe particular detection method.

An aspect of the present invention relates to the use of a nucleic acidmolecule as described herein as a probe, wherein the probe is anucleotide sequence selected from the group consisting of SEQ ID NOs: 1to 29, and/or a nucleotide sequence which is complementary to any of thenucleotide sequences in the group consisting of SEQ ID NOs: 1 to 29, ora fragment hereof, can hybridize under stringent condition and/or has anidentity of at least 80% to any of these sequences. In a preferredembodiment the probe is selected from the group of SEQ ID NOs: 1, 5, 6,7, 8, 9, 13, 14, 15, 16, 19 and 29 and/or a nucleotide sequence which iscomplementary to any of the nucleotide sequences in the group consistingof SEQ ID NOs: 1 to 29, or a fragment hereof, can hybridize understringent conditions and/or has an identity of at least 80% to any ofthese sequences. Most preferably the probe is either SEQ ID NO: 1, 9, 19or 29 and/or a nucleotide sequence which is complementary to either SEQID NO: 1, 9, 19 or 29 and/or a fragment hereof, and/or can hybridizeunder stringent condition and/or has an identity of at least 80% to anyof these sequences. Any of the herein described probes may be modifiedby labelling or immobilization as mentioned above.

Detection Methods

An aspect of the present invention regards the detection of nucleic acidmolecules by any method known in the art. In the following are givenexamples of various detection methods that can be employed for thispurpose, and the present invention includes all the mentioned methods,but is not limited to any of these.

In Situ Hybridization

In situ hybridization (ISH) applies and extrapolates the technology ofnucleic acid hybridization to the single cell level, and, in combinationwith the art of cytochemistry, immunocytochemistry andimmunohistochemistry, permits the maintenance of morphology and theidentification of cellular markers to be maintained and identified,allows the localization of sequences to specific cells withinpopulations, such as tissues and blood samples. ISH is a type ofhybridization that uses a complementary nucleic acid to localize one ormore specific nucleic acid sequences in a portion or section of tissue(in situ), or, if the tissue is small enough, in the entire tissue(whole mount ISH). DNA ISH can be used to determine the structure ofchromosomes and the localization of individual genes and optionallytheir copy numbers. Fluorescent DNA ISH (FISH) can for example be usedin medical diagnostics to assess chromosomal integrity. RNA ISH is usedto assay expression and gene expression patterns in a tissue/acrosscells, such as the expression of miRNAs/nucleic acid molecules as hereindescribed.

Sample cells are treated to increase their permeability to allow theprobe to enter the cells, the probe is added to the treated cells,allowed to hybridize at pertinent temperature, and then excess probe iswashed away. A complementary probe is labelled with a radioactive,fluorescent or antigenic tag, so that the probe's location and quantityin the tissue can be determined using autoradiography, fluorescencemicroscopy or immunoassay, respectively. The sample may be any sample asherein described and will often be a FFPE sample. The probe is likewisea probe according to any probe mentioned herein. An example of themethod of detection of selected miRNAs by the method of in situhybridization is given in Example 2.

An embodiment of the present invention regards the method of detectionby in situ hybridization as described herein.

In Situ PCR

In situ PCR is the PCR based amplification of the target nucleic acidsequences prior to ISH. For detection of RNA, an intracellular reversetranscription (RT) step is introduced to generate complementary DNA fromRNA templates prior to in situ PCR. This enables detection of low copyRNA sequences.

Prior to in situ PCR, cells or tissue samples are fixed andpermeabilized to preserve morphology and permit access of the PCRreagents to the intracellular sequences to be amplified. PCRamplification of target sequences is next performed either in intactcells held in suspension or directly in cytocentrifuge preparations ortissue sections on glass slides. In the former approach, fixed cellssuspended in the PCR reaction mixture are thermally cycled usingconventional thermal cyclers. After PCR the cells are cytocentrifugatedonto glass slides with visualization of intracellular PCR products byISH or immunohistochemistry. In situ PCR on glass slides is performed byoverlaying the samples with the PCR mixture under a coverslip which isthen sealed to prevent evaporation of the reaction mixture. Thermalcycling is achieved by placing the glass slides either directly on topof the heating block of a conventional or specially designed thermalcycler or by using thermal cycling ovens. Detection of intracellularPCR-products is achieved by one of two entirely different techniques. Inindirect in situ PCR by ISH with PCR-product specific probes, or indirect in situ PCR without ISH through direct detection of labellednucleotides (e.g. digoxigenin-11-dUTP, fluorescein-dUTP, 3H-CTP orbiotin-16-dUTP) which have been incorporated into the PCR productsduring thermal cycling.

An embodiment of the present invention regards the method of in situ PCRas mentioned herein above for the detection of nucleic acid molecules asdetailed herein.

Microarray

A microarray is a microscopic, ordered array of nucleic acids, proteins,small molecules, cells or other substances that enables parallelanalysis of complex biochemical samples. A DNA microarray consists ofdifferent nucleic acid probes, known as capture probes that arechemically attached to a solid substrate, which can be a microchip, aglass slide or a microsphere-sized bead. Microarrays can be used e.g. tomeasure the expression levels of large numbers of mRNAs/miRNAssimultaneously.

Microarrays can be fabricated using a variety of technologies, includingprinting with fine-pointed pins onto glass slides, photolithographyusing pre-made masks, photolithography using dynamic micromirrordevices, ink-jet printing, or electrochemistry on microelectrode arrays.

An aspect of the present invention regards the use of microarrays forthe expression profiling of miRNAs in hyperproliferative diseases. Forthis purpose, RNA is extracted from a cell or tissue sample, the smallRNAs (18-26-nucleotide RNAs) are size-selected from total RNA usingdenaturing polyacrylamide gel electrophoresis (PAGE). Thenoligonucleotide linkers are attached to the 5′ and 3′ ends of the smallRNAs and the resulting ligation products are used as templates for anRT-PCR reaction with 10 cycles of amplification. The sense strand PCRprimer has a Cy3 fluorophore attached to its 5′ end, therebyfluorescently labelling the sense strand of the PCR product. The PCRproduct is denatured and then hybridized to the microarray. A PCRproduct, referred to as the target nucleic acid that is complementary tothe corresponding miRNA capture probe sequence on the array willhybridize, via base pairing, to the spot at which the capture probes areaffixed. The spot will then fluoresce when excited using a microarraylaser scanner. The fluorescence intensity of each spot is then evaluatedin terms of the number of copies of a particular miRNA, using a numberof positive and negative controls and array data normalization methods,which will result in assessment of the level of expression of aparticular miRNA.

Alternatively, total RNA containing the small RNA fraction (includingthe miRNA) extracted from a cell or tissue sample is used directlywithout size-selection of small RNAs, and 3′ end labeled using T4 RNAligase and either a Cy3- or Cy5-labeled short RNA linker (f. ex.5″-PO4-rUrUrU-Cy3/dT-3′ or 5″-PO4-rUrUrU-Cy5/dT-3′). The RNA samples arelabelled by incubation at 30° C. for 2 hours followed by heatinactivation of the T4 RNA ligase at 80° C. for 5 minutes. Thefluorophore-labelled miRNAs complementary to the corresponding miRNAcapture probe sequences on the array will hybridize, via base pairing,to the spot at which the capture probes are affixed. The microarrayscanning and data processing is carried out as above.

Several types of microarrays can be employed such as spottedoligonucleotide microarrays, pre-fabricated oligonucleotide microarraysor spotted long oligonucleotide arrays

In spotted oligonucleotide microarrays the capture probes areoligonucleotides complementary to miRNA sequences. This type of array istypically hybridized with amplified PCR products of size-selected smallRNAs from two samples to be compared (e.g. hyperproliferative and normalsamples from an individual) that are labelled with two differentfluorophores. Alternatively, total RNA containing the small RNA fraction(including the miRNAs) is extracted from the abovementioned two samplesand used directly without size-selection of small RNAs, and 3′ endlabeled using T4 RNA ligase and short RNA linkers labelled with twodifferent fluorophores.

The samples can be mixed and hybridized to one single microarray that isthen scanned, allowing the visualization of up-regulated anddown-regulated miRNA genes in one go. The downside of this is that theabsolute levels of gene expression cannot be observed, but the cost ofthe experiment is reduced by half. Alternatively, a universal referencecan be used, comprising of a large set of fluorophore-labelledoligonucleotides, complementary to the array capture probes.

In pre-fabricated oligonucleotide microarrays or single-channelmicroarrays, the probes are designed to match the sequences of known orpredicted miRNAs. There are commercially available designs that covercomplete genomes from companies such as Affymetrix, or Agilent. Thesemicroarrays give estimations of the absolute value of gene expressionand therefore the comparison of two conditions requires the use of twoseparate microarrays.

Spotted long Oligonucleotide Arrays are composed of 50 to 70-meroligonucleotide capture probes, and are produced by either ink-jet orrobotic printing. Short Oligonucleotide Arrays are composed of 20-25-meroligonucleotide probes, and are produced by photolithographic synthesis(Affymetrix) or by robotic printing. More recently, Maskless ArraySynthesis from NimbleGen Systems has combined flexibility with largenumbers of probes. Arrays can contain up to 390,000 spots, from a customarray design.

A preferred embodiment of the present invention regards the method ofmicroarray use and analysis as described herein.

A more preferred embodiment of the present invention regards the use ofmicroarrays for the expression profiling of miRNAs in hyperproliferativediseases such as cancer and especially breast cancer.

A most preferred embodiment of the present invention regards the use ofmicroarrays for the expression profiling of miRNAs such as any of SEQ IDNO: 1 to 29 and especially SEQ ID NO 1 and/or 9 and/or 19 and/or 29 inhyperproliferative diseases such as cancer and especially breast andlung cancers.

PCR

The terms “PCR reaction”, “PCR amplification”, “PCR”, “pre-PCR”,“Q-PCR”, “real-time quantitative PCR” and “real-time quantitativeRT-PCR” are interchangeable terms used to signify use of a nucleic acidamplification system, which multiplies the target nucleic acids beingdetected. Examples of such systems include the polymerase chain reaction(PCR) system and the ligase chain reaction (LCR) system. Other methodsrecently described and known to the person of skill in the art are thenucleic acid sequence based amplification and Q Beta Replicase systems.The products formed by said amplification reaction may or may not bemonitored in real time or only after the reaction as an end-pointmeasurement.

Real-Time Quantitative RT-PCR

Real-time quantitative RT-PCR is a modification of polymerase chainreaction used to rapidly measure the quantity of a product of polymerasechain reaction. It is preferably done in real-time, thus it is anindirect method for quantitatively measuring starting amounts of DNA,complementary DNA or ribonucleic acid (RNA). This is commonly used forthe purpose of determining whether a genetic sequence is present or not,and if it is present the number of copies in the sample. There are 3methods which vary in difficulty and detail. Like other forms ofpolymerase chain reaction, the process is used to amplify DNA samples,using thermal cycling and a thermostable DNA polymerase.

The three commonly used methods of quantitative polymerase chainreaction are through agarose gel electrophoresis, the use of SYBR Green,a double stranded DNA dye, and the fluorescent reporter probe. Thelatter two of these three can be analysed in real-time, constitutingreal-time polymerase chain reaction method.

Agarose gel electrophoresis is the simplest method, but also often slowand less accurate then other methods, depending on the running of anagarose gel via electrophoresis. It cannot give results in real time.The unknown sample and a known sample are prepared with a knownconcentration of a similarly sized section of target DNA foramplification. Both reactions are run for the same length of time inidentical conditions (preferably using the same primers, or at leastprimers of similar annealing temperatures). Agarose gel electrophoresisis used to separate the products of the reaction from their original DNAand spare primers. The relative quantities of the known and unknownsamples are measured to determine the quantity of the unknown. Thismethod is generally used as a simple measure of whether the probe targetsequences are present or not, and rarely as ‘true’ Q-PCR.

Using SYBR Green dye is more accurate than the gel method, and givesresults in real time. A DNA binding dye binds all newly synthesizeddouble stranded (ds)DNA and an increase in fluorescence intensity ismeasured, thus allowing initial concentrations to be determined.However, SYBR Green will label all dsDNA including any unexpected PCRproducts as well as primer dimers, leading to potential complicationsand artefacts. The reaction is prepared as usual, with the addition offluorescent dsDNA dye. The reaction is run, and the levels offluorescence are monitored; the dye only fluoresces when bound to thedsDNA. With reference to a standard sample or a standard curve, thedsDNA concentration in the PCR can be determined.

The fluorescent reporter probe method is the most accurate and mostreliable of the methods. It uses a sequence-specific nucleic acid basedprobe so as to only quantify the probe sequence and not all doublestranded DNA. It is commonly carried out with DNA based probes with afluorescent reporter and a quencher held in adjacent positions,so-called dual-labelled probes. The close proximity of the reporter tothe quencher prevents its fluorescence; it is only on the breakdown ofthe probe that the fluorescence is detected. This process depends on the5′ to 3′ exonuclease activity of the polymerase involved. The real-timequantitative PCR reaction is prepared with the addition of thedual-labelled probe. On denaturation of the double-stranded DNAtemplate, the probe is able to bind to its complementary sequence in theregion of interest of the template DNA (as the primers will too). Whenthe PCR reaction mixture is heated to activate the polymerase, thepolymerase starts synthesizing the complementary strand to the primedsingle stranded template DNA. As the polymerisation continues it reachesthe probe bound to its complementary sequence, which is then hydrolyseddue to the 5′-3′ exonuclease activity of the polymerase therebyseparating the fluorescent reporter and the quencher molecules. Thisresults in an increase in fluorescence, which is detected. Duringthermal cycling of the real-time PCR reaction, the increase influorescence, as released from the hydrolysed dual-labelled probe ineach PCR cycle is monitored, which allows accurate determination of thefinal, and so initial, quantities of DNA.

Any method of PCR that can determine the expression of a nucleic acidmolecule as defined herein falls within the scope of the presentinvention. A preferred embodiment of the present invention regards thereal-time quantitative RT-PCR method, based on the use of either SYBRGreen dye or a dual-labelled probe for the detection and quantificationof nucleic acids according to the herein described. Examples of methodsfor detection of nucleic acid molecules are given in Example 4 and 5

A more preferred embodiment of the present invention regards the methodsof real-time quantitative RT-PCR for the expression profiling of miRNAsin hyperproliferative diseases such as cancer and especially breastcancer.

A most preferred embodiment of the present invention regards the methodsof real-time quantitative RT-PCR for the expression profiling of miRNAssuch as any of SEQ ID NO: 1 to 29 and especially SEQ ID NO 1 and/or 9and/or 19 and/or 29 in hyperproliferative diseases such as cancer andespecially breast and lung cancers.

Northern Blot Analysis

An aspect of the present invention regards the detection of the nucleicacid molecules herein disclosed by the classical and to the artwell-known technique of Northern blot analysis. Many variations of theprotocol exist and optimizations regarding the detection of miRNAsconstitute preferred embodiments of the present invention. It has beenindicated that a critical factor in detecting miRNAs is the membrane towhich the RNA being detected is bound. An example of the method ofNorthern blot analysis is given in Example 1.

Affinity Matrices

Affinity matrices may be used as a sample preparation method orpreferably as a method of detection. The type of affinity matrix useddepends on the purpose of the analysis. For example, where it is desiredto analyse miRNA expression levels of particular genes in a complexnucleic acid sample it is often desirable to eliminate ribonucleic acidsproduced by genes that are constitutively overexpressed and thereby tendto mask gene products expressed at characteristically lower levels.Thus, in one embodiment, the affinity matrix can be used to remove anumber of preselected gene products (e.g., actin, GAPDH, globin mRNAs ina blood sample etc.). Similarly, the affinity matrix can be used toefficiently capture, i.e. isolate/detect, a number of known nucleic acidsequences. In this embodiment the matrix is also prepared bearingnucleic acids complementary to those nucleic acids it is desired toisolate. The sample is contacted to the matrix under conditions wherethe complementary nucleic acid sequences hybridize to the affinityligands in the matrix. The non-hybridized material is washed off thematrix leaving the desired sequences bound. The hybrid duplexes are thendenatured providing a pool of the isolated nucleic acids. The differentnucleic acids in the pool can be subsequently separated according tostandard methods e.g. gel electrophoresis.

In another embodiment, the affinity matrix is a bead, e.g. acarboxylated five micron polystyrene bead. 5′ amino-linedoligonucleotide capture probes complementary to the miRNAs of interestare coupled to the beads impregnated with variable mixtures of twofluorescent dyes that can yield up to 100 colours, each representing asingle miRNA species. RNA is isolated from the sample, small RNAs arefractionated, and then RNA oligonucleotide linkers are attached to the5′ and 3′ ends of the small RNAs. The resulting ligation products areamplified by polymerase chain reaction (PCR) using a common biotinylatedprimer, hybridized to the capture beads, and stained withstreptavidin-phycoerythrin. The beads are then analysed using a flowcytometer capable of measuring bead colour (denoting miRNA identity) andphycoerythrin intensity (denoting miRNA abundance).

Alternatively, 5′.amino-linked LNA-modified capture probes complementaryto the 5′-end of the miRNAs of interest are coupled to the colour-codedbeads, contacted with the sample of interest, e.g. a total RNA sample,followed by hybridization of another LNA-modified probe, a so-calleddetection probe containing a biotin or a fluorophore, which saiddetection probe hybridizes to the 3′-end of the miRNA of interest. Thebeads are analysed using a flow cytometer measuring bead colour(denoting miRNA identity) and fluorescence intensity from the detectionprobe (denoting miRNA abundance).

In a preferred embodiment the abovementioned matrix-based detectionmethods are applied to the Luminex xMap technology platform and theLuminex compact analyser.

A more preferred embodiment of the present invention regards the methodsof matrix-based detection for the expression profiling of miRNAs inhyperproliferative diseases such as cancer and especially breast cancer.

A most preferred embodiment of the present invention regards the methodsof matrix-based detection for the expression profiling of miRNAs such asany of SEQ ID NO: 1 to 29 and especially SEQ ID NO 1 and/or 9 and/or 19and/or 29 in hyperproliferative diseases such as cancer and especiallybreast and lung cancers.

An embodiment of the present invention comprises any of the hereinabovementioned methods for the detection of nucleic acid molecules. In apreferred embodiment an in situ detection method is employed. In anotherpreferred embodiment a screening method is employed. In a more preferredembodiment a method of real-time PCR or microarray analysis is employed.

Preferably, an embodiment of the invention comprises a method ofdetecting any of the miRNAs of SEQ ID NO: 1 to 29. More preferably, anembodiment of the invention comprises a method of detecting any of themiRNAs of SEQ ID NO: 1, 3, 4, 5, 6, 7, 8, 9, 13, 14, 15, 16, 19, 20, 21,22, 23, 24, 25, 26, 27, 28 and 29. Most preferably, an embodiment of theinvention comprises a method of detecting any of the miRNAs of SEQ IDNO: 1, 9, 19, 25 or 29.

In a further preferred embodiment the invention comprises a method fordetecting miR-31 (miRNA of SEQ ID NO: 25) expression, in particular amethod detecting an increase level of expression of miR-31, wherein theexpression level of miR-31 is higher than a standard value or higherthan in corresponding healthy tissue. Such a method may involvedetection of miR-31 expression by in situ hybridization, array analysisor PCR, preferably real-time PCR analysis.

Pharmaceutical Composition

“Pharmaceutical agent, drug or composition” refers to any chemical orbiological material, compound, or composition capable of inducing adesired prophylactic or therapeutic effect when properly administered toa patient. Some drugs are sold in an inactive form that is converted invivo into a metabolite with pharmaceutical activity. For purposes of thepresent invention, the terms “pharmaceutical agent, drug or composition”encompass both the inactive drug and the active metabolite or derivate.Specifically the pharmaceutical agent is an agent with a miRNA targetingmoiety. The agent may regulate expression/activity or otherwiseinterfere with the function of a miRNA and/or pre-miRNA.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration and may thuscomprise a pharmaceutically acceptable carrier. Examples of routes ofadministration include parenteral, e.g., intravenous, intradermal,subcutaneous, intraperitoneal, intramuscular, oral (e.g., inhalation),transdermal (topical), pulmonary and transmucosal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylene-diamine-tetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

It is an aspect of the present invention that the administration form isspecifically suited for the disease/disorder to be treated. For example,parenteral and pulmonary administration forms of the active ingredient(e.g. an agent with a miRNA targeting moiety) may be used in thetreatment of disorders/diseases of the lungs.

The compounds of the present invention may be formulated for parenteraladministration (e.g., by injection, for example bolus injection orcontinuous infusion) and may be presented in unit dose form in ampoules,pre-filled syringes, small volume infusion or in multi-dose containerswith an added preservative. The compositions may take such forms assuspensions, solutions, or emulsions in oily or aqueous vehicles, forexample solutions in aqueous polyethylene glycol. Examples of oily ornonaqueous carriers, diluents, solvents or vehicles include propyleneglycol, polyethylene glycol, vegetable oils (e.g., olive oil), andinjectable organic esters (e.g., ethyl oleate), and may containformulatory agents such as preserving, wetting, emulsifying orsuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient may be in powder form, obtained by aseptic isolationof sterile solid or by lyophilisation from solution for constitutionbefore use with a suitable vehicle, e.g., sterile, pyrogen-free water.

Oils useful in parenteral formulations include petroleum, animal,vegetable, or synthetic oils. Specific examples of oils useful in suchformulations include peanut, soybean, sesame, cottonseed, corn, olive,petrolatum, and mineral. Suitable fatty acids for use in parenteralformulations include oleic acid, stearic acid, and isostearic acid.Ethyl oleate and isopropyl myristate are examples of suitable fatty acidesters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides; (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example,alkyl-.beta.-aminopropionates, and 2-alkyl-imidazoline quaternaryammonium salts, and (e) mixtures thereof.

The parenteral formulations typically will contain from about 0.5 toabout 25% by weight of the active ingredient in solution. Preservativesand buffers may be used. In order to minimize or eliminate irritation atthe site of injection, such compositions may contain one or morenonionic surfactants having a hydrophile-lipophile balance (HLB) of fromabout 12 to about 17. The quantity of surfactant in such formulationswill typically range from about 5 to about 15% by weight. Suitablesurfactants include polyethylene sorbitan fatty acid esters, such assorbitan monooleate and the high molecular weight adducts of ethyleneoxide with a hydrophobic base, formed by the condensation of propyleneoxide with propylene glycol. The parenteral formulations can bepresented in unit-dose or multi-dose sealed containers, such as ampulesand vials, and can be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid excipient, forexample, water, for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described.

For pulmonary administration, the compounds of the present invention maybe formulated for aerosol administration, particularly to therespiratory tract and including intranasal administration. The compoundwill generally have a small particle size for example of the order of 5microns or less. Such a particle size may be obtained by means known inthe art, for example by micronization. The active ingredient is providedin a pressurized pack with a suitable propellant such as achlorofluorocarbon (CFC) for example dichlorodifluoromethane,trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide orother suitable gas. The aerosol may conveniently also contain asurfactant such as lecithin. The dose of drug may be controlled by ametered valve. Alternatively the active ingredients may be provided in aform of a dry powder, for example a powder mix of the compound in asuitable powder base such as lactose, starch, starch derivatives such ashydroxypropylmethyl cellulose and polyvinylpyrrolidine (PVP). The powdercarrier will form a gel in the nasal cavity. The powder composition maybe presented in unit dose form for example in capsules or cartridges ofe.g., gelatin or blister packs from which the powder may be administeredby means of an inhaler. Pulmonary lavage is a further administrationform that may be employed.

When desired, formulations can be prepared with enteric coatings adaptedfor sustained or controlled release administration of the activeingredient.

Pharmaceutically Acceptable Salts

Pharmaceutically acceptable salts of the instant compounds, where theycan be prepared, are also intended to be covered by this invention.These salts will be ones which are acceptable in their application to apharmaceutical use. By that it is meant that the salt will retain thebiological activity of the parent compound and the salt will not haveuntoward or deleterious effects in its application and use in treatingdiseases.

Pharmaceutically acceptable salts are prepared in a standard manner. Ifthe parent compound is a base it is treated with an excess of an organicor inorganic acid in a suitable solvent. If the parent compound is anacid, it is treated with an inorganic or organic base in a suitablesolvent.

The compounds of the invention may be administered in the form of analkali metal or earth alkali metal salt thereof, concurrently,simultaneously, or together with a pharmaceutically acceptable carrieror diluent, especially and preferably in the form of a pharmaceuticalcomposition thereof, whether by oral, rectal, or parenteral (includingsubcutaneous) route, in an effective amount.

Examples of pharmaceutically acceptable acid addition salts for use inthe present inventive pharmaceutical composition include those derivedfrom mineral acids, such as hydrochloric, hydrobromic, phosphoric,metaphosphoric, nitric and sulfuric acids, and organic acids, such astartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic,gluconic, succinic, p-toluenesulphonic acids, and arylsulphonic, forexample.

Pharmaceutical agent or drug” refers to any chemical or biologicalmaterial, compound, or composition capable of inducing a desiredtherapeutic effect when properly administered to a patient. Some drugsare sold in an inactive form that is converted in vivo into a metabolitewith pharmaceutical activity. For purposes of the present invention, theterms “pharmaceutical agent” and “drug” encompass both the inactive drugand the active metabolite.

“Transport” and “delivery” refers to the passage of a substance acrossor through the skin (i.e., transdermal), including the epidermis anddermis, or across a mucosal membrane (i.e., gastrointestinal,sublingual, buccal, nasal, pulmonary, vaginal, corneal, and ocularmembranes), where the substance can contact, and be absorbed into, thecapillaries. In certain instances, the delivery and/or transport of thesubstance across other membranes will be effected.

“Penetration enhancer” refers to a substance which is used to increasethe transdermal or transmembrane flux of a compound. A penetrationenhancer is typically applied to the skin or mucous membrane incombination with the compound. Enhancers are believed to function bydisrupting the skin or mucous membrane barrier or changing thepartitioning behavior of the drug in the skin or mucous membrane.

The use and administration of the herein disclosed pharmaceuticalcomponents in combination with the pharmaceutically active agent asherein described is standard practice by those skilled in the art.

A therapeutically effective amount of a composition containing acomposition of the invention is an amount that is capable of modulatingthe expression of a pre-miRNA, miRNA or the target of a miRNA accordingto the herein described. Certain factors may influence the dosage andtiming required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of a composition can include a single treatment or aseries of treatments.

In relation to cancer diseases, upregulated miRNAs are particularlyinteresting, as potential Onco-miRNA's. Such Onco-miRNA's may act bydown-regulating expression of tumor suppressors providing a strongproliferative stimulation. MicroRNAs may target expression of more thanone gene, such as two genes or 3 or 4 genes or even such as 10 or 20genes. In a preferred embodiment of the invention the pharmaceuticalcomposition modulates expression of a pre-miRNA or miRNA that regulatesexpression of more than one gene, such as at least two genes. It isfurther preferred that the genes regulated by the miRNA are involved ingrowth regulation, most preferred are tumor suppressors.

In a preferred embodiment of the invention the pharmaceuticalcomposition comprises an agent that has a miRNA targeting moiety whichdown-regulates expression of one or more of the miRNAs or pre-miRNAs ofSEQ ID NO 4-8, 12-18 and 25-28. In a particularly preferred embodimentthe pharmaceutical composition comprises an agent which downregulatesexpression of miRNA of SEQ ID NO 25.

In a further preferred embodiment of the invention, the pharmaceuticalcomposition comprises an agent that has a miRNA targeting moiety whichupregulates expression of the miRNAs or pre-miRNAs of SEQ ID NO 1-3,9-11, 19-24 and 29.

In an embodiment, the agent that has a miRNA targeting moiety, is anucleic acid comprising:

-   -   a) the nucleotide sequence of SEQ ID NO: 4-8, 12-18 and 25-28        and/or,    -   b) a nucleotide sequence which is complementary to a) and/or,    -   c) a nucleotide sequence which is a fragment of a) or b) and/or,    -   d) a nucleotide sequence which has an identity of at least 80%        to a sequence of a), b) or c) and/or,    -   e) a nucleotide sequence which hybridizes under stringent        conditions to a sequence of a), b), c) or d)

In a preferred embodiment the agent is:

-   -   b) a nucleotide sequence which is complementary to the        nucleotide sequence of SEQ ID NO: 4-8, 12-18 and 25-28 and/or,    -   c) a nucleotide sequence which is a fragment of b) and/or,    -   d) a nucleotide sequence which has an identity of at least 80%        to a sequence of b) and/or    -   e) a nucleotide sequence which hybridizes under stringent        conditions to a sequence of b).

In a more preferred embodiment the agent is

-   -   b) a nucleotide sequence which is complementary to the        nucleotide sequence of SEQ ID NO: 25 and/or    -   c) a nucleotide sequence which is a fragment of b) and/or,    -   d) a nucleotide sequence which has an identity of at least 80%        to a sequence of b) and/or    -   e) a nucleotide sequence which hybridizes under stringent        conditions to a sequence of b).

It is clear from the above that an agent according to the invention maybe comprised by a composition, a nucleic acid construct, a deliveryvehicle, a cell, a kit or a pharmaceutical composition.

Second Active Ingredient

An aspect of the present invention regards the pharmaceuticalcomposition according to any of the herein disclosed comprising a secondactive ingredient. The term second active ingredient includes anytherapeutic molecule or cocktail of molecules other than the at leastone nucleic acid molecule of the herein described.

An embodiment of the present invention regards the prophylaxis andtherapy of diseases such as hyperproliferative diseases e.g. cancer andespecially breast cancer. It is thus an object of the present inventionto provide a pharmaceutical composition comprising a second activeingredient that may be beneficial, i.e. function synergistically, inthis regards.

A second active ingredient may therefore be any therapeutic moleculethat may be used prophylactically or therapeutically in the treatment ofhyperproliferative diseases such as cancer and especially breast cancer.Such therapeutic molecules include, but are not limited to,chemotherapeutic agents, anti-emetic drugs, anti-inflammatory agents,anti-allergenics, cytokines and antibiotics.

Chemotherapy is the use of chemical substances to treat disease. In itsmodern-day use, it refers primarily to cytotoxic drugs used to treatcancer. The majority of chemotherapeutic drugs can be divided in to:alkylating agents, antimetabolites, anthracyclines, plant alkaloids,topoisomerase inhibitors, and antitumour agents. All of these drugsaffect cell division or DNA synthesis and function in some way. Somenewer agents don't directly interfere with DNA. These include the newtyrosine kinase inhibitor imatinib mesylate, which directly targets amolecular abnormality in certain types of cancer (chronic myelogenousleukaemia, gastrointestinal stromal tumours). In addition, some drugsmay be used which modulate tumour cell behaviour without directlyattacking those cells. Hormonal therapy falls into this category.

Several malignancies respond to hormonal therapy. Cancer arising fromcertain tissues including the mammary glands may be inhibited orstimulated by appropriate changes in hormone balance. Steroids (oftendexamethasone) can inhibit tumour growth or the associated edema (tissueswelling), and may cause regression of lymph node malignancies. Breastcancer cells often highly express the estrogen and/or progesteronereceptor. Inhibiting the production (with aromatase inhibitors) oraction (with tamoxifen) of these hormones can often be used as anadjunct to therapy. Gonadotropin-releasing hormone agonists (GnRH), suchas goserelin possess a paradoxic negative feedback effect followed byinhibition of the release of FSH (follicle-stimulating hormone) and LH(luteinizing hormone), when given continuously.

A preferred embodiment of the present invention compriseschemotherapeutic agents such as antineoplastic agents, radioiodinatedcompounds, toxins, cytostatic and cytolytic drugs, alkylating agents,anti-metabolites, anthracyclines, plant alkaloids, topoisomeraseinhibitors, anti-tumour agents, tyrosine kinase inhibitors and hormoneagents.

A more preferred embodiment of the present invention compriseschemotherapeutic agents such as any of the above specifically for usewith breast cancer. Examples of these include, but are not limited to:Cyclophosphamide, Epirubicin, 5-Fluorouracil or 5 FU, Methotrexate,Mitomycin, Mitozantrone (mitoxantrone), Doxorubicin (Adriamycin),Herceptin, Tamoxifen and other aromatase inhibitors.

These drugs are given in different combinations, also called cocktails.Some of the commonest combinations used for breast cancer areCMF—cyclophosphamide, methotrexate and 5-FU; FEC—epirubicin,cyclophosphamide and 5-FU; E-CMF—epirubicin, followed by CMF;AC—doxorubicin (adriamycin) and cyclophosphamide; MMM—methotrexate,mitozantrone and mitomycin; MM—methotrexate and mitozantrone. All ofthese fall within the scope of the present invention.

Kit of Parts

An aspect of the present invention regards the inclusion of nucleic acidmolecules of the invention in a container, pack, kit or dispensertogether with instructions for administration or use. The kit may be forthe detection of a nucleic acid molecule, or for the classification,diagnosis and/or prognosis of a disease related to the nucleic acidmolecule such as a hyperproliferative disease, e.g. a cancer especiallybreast cancer. The kit may furthermore provide compositions comprisingat least one nucleic acid molecule of the invention for the treatment ofdiseases such those mentioned herein.

The kit can comprise the nucleic acid molecules or probes of theinvention in a form suitable for the detection of said nucleic acidmolecules. Thus the kit may comprise the nucleic acid molecules in anycomposition suitable for the use of the nucleic acid molecules accordingto the instructions. Furthermore, the kit may comprise any detectiondevice required here for, such as a microarray, a labelling system, acocktail of components e.g. suspensions required for any type of PCR,especially real-time quantitative RT-PCR, membranes, colour-coded beads,columns or other.

The kit may furthermore contain a pharmaceutical composition comprisingthe nucleic acid of the invention and an instructional material for theprophylaxis or treatment of a hyperproliferative disease. Such a kit mayinclude a container having the pharmaceutical composition of theinvention. Further, a kit comprising a pharmaceutical composition and adelivery device for delivering the composition to an individual can alsobe provided. By way of example, the delivery device may be an aerosolspray device, an atomizer, a dry powder delivery device, aself-propelling solvent/powder-dispensing device, a syringe, a needle,or a dosage measuring container.

An embodiment of the present invention thus regards pharmaceuticalcompositions comprising nucleic acid molecules as herein described.Especially pharmaceutical compositions comprising nucleic acid moleculesaccording to SEQ ID NO 1 or 9 or derivates as defined herein arepreferred.

Vehicles

Aspects of the present invention relate to various vehicles comprisingthe nucleic acid molecules of the present invention. By vehicle isunderstood an agent with which genetic material can be transferred.Herein such vehicles are exemplified as nucleic acid constructs,vectors, and delivery vehicles such as viruses and cells.

Nucleic Acid Construct

By nucleic acid construct is understood a genetically engineered nucleicacid. The nucleic acid construct may be a non-replicating and linearnucleic acid, a circular expression vector, an autonomously replicatingplasmid or viral expression vector. A nucleic acid construct maycomprise several elements such as, but not limited to genes or fragmentsof same, promoters, enhancers, terminators, poly-A tails, linkers,markers and host homologous sequences for integration. Methods forengineering nucleic acid constructs are well known in the art (see,e.g., Molecular Cloning: A Laboratory Manual, Sambrook et al., eds.,Cold Spring Harbor Laboratory, 2nd Edition, Cold Spring Harbor, N.Y.,1989).

An embodiment of the invention comprises the at least one nucleic acidmolecule as herein described comprised within a nucleic acid constructaccording to the above. Preferred embodiments are nucleic acidconstructs comprising at least one of SEQ ID NOs 1 to 29 or theircomplement, and more preferred embodiments are nucleic acid constructscomprising at least one of SEQ ID NOs 1, 9, 19, 25 or 29 or theircomplement.

Several nucleic acid molecules may be encoded within the same constructand may be linked by an operative linker. By the term operative linkeris understood a sequence of nucleotides two parts of a nucleic acidconstruct in a manner securing the biological processing of the encodednucleic acid molecules.

Promoter

The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator proteins. At least one module ineach promoter functions to position the start site for RNA synthesis.The best known example of this is the TATA box, but in some promoterslacking a TATA box, such as the promoter for the mammalian terminaldeoxynucleotidyl transferase gene and the promoter for the SV 40 lategenes, a discrete element overlying the start site itself helps to fixthe place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. Depending on the promoter, it appears that individual elementscan function either cooperatively or independently to activatetranscription. Any promoter that can direct transcription initiation ofthe sequences encoded by the nucleic acid construct may be used in theinvention.

An aspect of the present invention comprises the nucleic acid constructwherein the sequence of at least one nucleic acid molecule is precededby a promoter enabling expression of the at least one nucleic acidmolecule. This nucleic acid molecule preferably being at least one ofSEQ ID NO: 1 to 29, and more preferably being at least one of SEQ ID NO1, 9, 19 or 29.

It is a further aspect that the promoter is selected from the group ofconstitutive promoters, inducible promoters, organism specificpromoters, tissue specific promoters and cell type specific promoters.Examples of promoters include, but are not limited to: constitutivepromoters such as: simian virus 40 (SV40) early promoter, a mousemammary tumour virus promoter, a human immunodeficiency virus longterminal repeat promoter, a Moloney virus promoter, an avian leukaemiavirus promoter, an Epstein-Barr virus immediate early promoter, a Roussarcoma virus (RSV) promoter, a human actin promoter, a human myosinpromoter, a human haemoglobin promoter, cytomegalovirus (CMV) promoterand a human muscle creatine promoter, inducible promoters such as: ametallothionine promoter, a glucocorticoid promoter, a progesteronepromoter, and a tetracycline promoter (tet-on or tet-off), tissuespecific promoters such as: HER-2 promoter and PSA associated promoter.

Delivery Vehicle

An aspect of the present invention comprises the nucleic acid constructas described in any of the above, comprised within a delivery vehicle. Adelivery vehicle is an entity whereby a nucleotide sequence can betransported from at least one media to another. Delivery vehicles aregenerally used for expression of the sequences encoded within thenucleic acid construct and/or for the intracellular delivery of theconstruct. It is within the scope of the present invention that thedelivery vehicle is a vehicle selected from the group of: RNA basedvehicles, DNA based vehicles/vectors, lipid based vehicles, virallybased vehicles and cell based vehicles. Examples of such deliveryvehicles include, but are not limited to: biodegradable polymermicrospheres, lipid based formulations such as liposome carriers,coating the construct onto colloidal gold particles,lipopolysaccharides, polypeptides, polysaccharides, pegylation of viralvehicles.

A preferred embodiment of the present invention comprises a virus as adelivery vehicle, where the virus is selected from the non-exhaustivegroup of: adenoviruses, retroviruses, lentiviruses, adeno-associatedviruses, herpesviruses, vaccinia viruses, foamy viruses,cytomegaloviruses, Semliki forest virus, poxviruses, RNA virus vectorand DNA virus vector. Such viral vectors are well known in the art.

Recombinant Cell

An aspect of the present invention relates to a cell comprising thenucleic acid construct as defined in any of the above. Such arecombinant cell can be used a tool for in vitro research, as a deliveryvehicle for the nucleic acid construct or as part of a gene therapyregime. The nucleic acid construct and nucleic acid based vectorsaccording to the invention can be introduced into cells by techniqueswell known in the art and which include microinjection of DNA into thenucleus of a cell, transfection, electroporation, lipofection/liposomefusion and particle bombardment. Suitable cells include autologous andnon-autologous cells, and may include xenogenic cells.

An embodiment of the present invention is a pharmaceutical compositioncomprising any of the vehicles described herein, these vehicles beingnucleic acid constructs, vectors, delivery vehicles and or cells. Ofthese embodiments comprising any of SEQ ID NO 1 to 29 are preferred andmore preferred are embodiments comprising any of SEQ ID NO 1 and/or 9and/or 19 and/or 29.

Treatment

The present invention further provides for compositions and methods fortreating an individual having or at risk of acquiring a disease ordisorder. Treatment includes prophylaxis and/or therapy. The disease maybe characterized or caused by the overexpression or overactivity of agene product, or alternatively, may be caused by the expression oractivity of a mutant gene or gene product. The disease may thus be anyof the herein mentioned diseases such as hyperproliferative diseasessuch as cancer and especially breast cancer.

Accordingly, administration of an agent that has a miRNA targetingmoiety capable of binding said miRNA, being this either mature orprecursor miRNA, or an agent that has an miRNA target binding capabilityfalls within the scope of the present invention. The agent is preferablyany of the herein disclosed nucleic acid molecules.

By miRNA target is meant the target mRNA of the miRNA. The target mRNAsof some miRNAs are well characterized and described, for others, thetargets are predicted. It is predicted that each miRNA has multipletargets. For a list of predicted targets of miRNA-451 see Example 6.

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of or susceptible to, a disease orhaving a disease associated with aberrant or unwanted target geneexpression or activity. Another aspect of the invention pertains tomethods of modulating target gene expression, gene product expression oractivity for therapeutic purposes. Accordingly, in an exemplaryembodiment, the modulatory method of the invention involves contacting acell capable of expressing a target miRNA or the target mRNA of an miRNAwith a therapeutic agent (e.g. a nucleic acid molecule as describedherein) that is specific for the target gene or gene product (e.g. anucleic acid molecule as described herein) such that expression or oneor more of the activities of the target is modulated. These modulatorymethods can be performed in vitro (e.g., by culturing the cell with theagent) or, alternatively, in vivo (e.g., by administering the agent to asubject). As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant or unwanted expression or activity of a target gene product ornucleic acid molecule. Inhibition of target gene activity is desirablein situations in which the target gene is abnormally unregulated and/orin which decreased target gene activity is likely to have a beneficialeffect.

An embodiment of the present invention regards the use of any of theherein described pharmaceutical compositions for the treatment orprophylaxis of a disease such as a hyperproliferative disease, e.g.cancer especially breast cancer. A preferred embodiment of the presentinvention regards the treatment of breast cancers such as any of thebreast cancers known as: luminal A, luminal B, HER-2-overexpressing,basal and normal-like breast cancers.

A similarly preferred embodiment of the present invention regards thetreatment of lung cancers such as any of the lung cancers known as:small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC)

Yet an embodiment of the present invention regards the use of apharmaceutical composition for the modulation of expression or treatmentof a disease associated with any aberrant miRNA or any miRNA target mRNAexpression. Sequences of human miRNAs are collected in the miRbase.Targets of miRNAs are likewise found in miRbase, and it is an object ofthe present invention to treat or modulate the expression of targets ofany human miRNAs, preferably the herein mentioned miRNAs such as SEQ IDNOs 1 to 29 and most preferably SEQ ID NO 1, 9, 19 and 29.

An aspect of the present invention relates to the treatment of diseasescharacterized by the upregulation of miRNAs. Such treatments maycomprise administering nucleic acid molecules of the present inventionin order to downregulate the miRNAs and/or upregulate the targets ofsaid miRNAs. A preferred embodiment of the present invention relates totreatments counteracting the upregulation of hsa-miR-141, hsa-miR-200b,hsa-miR-200c, hsa-miR-221, has-miR-21, hsa-miR-222, hsa-miR-31,hsa-miR-127, hsa-miR-141, hsa-miR-136 and hsa-miR-376a when these areassociated with hyperproliferative diseases. Such treatment may comprisethe administration of inhibitory agents e.g. anti-sense molecules, todirectly interact with the overexpressed miRNAs.

Another preferred embodiment of the present invention relates to thetreatment of diseases characterized by the downregulation of miRNAs.Such treatments may comprise administering nucleic acid molecules of thepresent invention in order to upregulate the miRNAs and/or downregulatethe targets of said miRNAs. A preferred embodiment of the presentinvention relates to treatments counteracting the downregulation ofhsa-miR-451, hsa-miR-143, hsa-miR-145, hsa-miR-34b, hsa-miR-34c,hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-486, hsa-miR-451, hsa-miR-144and hsa-miR-150. Such treatment may comprise the administration nucleicacid molecules to supplement the lack of said miRNAs or inducer of theexpression of said miRNAs.

It is preferred that agents for regulation of expression of the miRNAsor pre-miRNAs in a cell, tissue or animal normalize the expression levelof the target miRNA or pre-miRNAs. It is further preferred that in caseof upregulated miRNAs the expression level is decreased by at least 10%,such as by 20%, such as by 30 or 50%, more preferably such as at least60% or 70%, most preferably at least 80 or 85%. In case of downregulatedmiRNAs it is preferred that the expression level is increased to atleast 10%, such as by 20%, such as by 30 or 50%, more preferably such asat least 60% or 70% most preferably such as at least 80, 90 or about100% of the normal expression level in corresponding healthy tissue.

As seen in FIGS. 8-16 upregulation of miR-31 was confirmed in bothmurine and human lung cancer cells. Directed downregulation of miR-31secondly conferred growth inhibition involving upregulation of LATS2 andPPP2R2A known as tumour suppressors.

Embodiments of the invention are further exemplified by the followingitems.

Items

-   -   1. A method for detection, classification, diagnosis or        prognosis of breast and/or lung cancer comprising the steps of:        -   a) obtaining at least one sample from an individual.        -   b) detecting the presence or absence of expression and/or            the expression level of at least one nucleic acid molecule,            said nucleic acid molecule comprising:        -   c) a nucleotide sequence selected from the group consisting            of SEQ ID NOs: 1, 5, 6, 7, 8, 9, 13, 14, 15, 16, 17, 18, in            relation to breast cancer and SEQ ID NOs: 1, 3, 4, 19, 20,            21, 22, 23, 24, 25, 26, 27, 28 and 29 in relation to lung            cancer.        -   d) a nucleotide sequence which is complementary to c)            and/or,        -   e) a nucleotide sequence which is a fragment of c) or d)            and/or,        -   f) a nucleotide sequence which has an identity of at least            80% to a sequence of c), d) or e) and/or,        -   g) a nucleotide sequence which hybridizes under stringent            conditions to a sequence of c), d), e) or f).    -   2. A method for detection, classification, diagnosis or        prognosis of a disease comprising the steps of:        -   a) obtaining at least one sample from an individual.        -   b) detecting the presence or absence of expression and/or            the expression level of at least one nucleic acid molecule,            said nucleic acid molecule comprising:        -   c) a nucleotide sequence selected from the group consisting            of SEQ ID NOs: 1, 3, 4, 9, 19, 20, 21, 22, 23, 24, 25, 26,            27, 28 and 29.        -   d) a nucleotide sequence which is complementary to c)            and/or,        -   e) a nucleotide sequence which is a fragment of c) or d)            and/or,        -   f) a nucleotide sequence which has an identity of at least            80% to a sequence of c), d) or e) and/or,        -   g) a nucleotide sequence which hybridizes under stringent            conditions to a sequence of c), d), e) or f).    -   3. The method of item 2, wherein the disease is a        hyperproliferative disease such as cancer.    -   4. The method according to item 3, wherein the cancer is breast        cancer.    -   5. The method according to item 3, wherein the cancer is lung        cancer.    -   6. The method according to any of items 1 to 5, comprising the        detection of the expression levels of at least two or more        nucleic acid molecules.    -   7. The method according to any of items 1 to 6, wherein the        nucleic acid molecule(s) are selected from the group of SEQ ID        NO: 1 to 29.    -   8. The method according to any of items 1 to 7, wherein at least        one nucleic acid molecule is SEQ ID NO: 1 or SEQ ID NO: 9 in        relation to breast cancer and/or SEQ ID NO: 19 or SEQ ID NO: 29        in relation to lung cancer.    -   9. The method according to any of items 1 to 8, wherein a probe        comprising:        -   a) a nucleotide sequence selected from the group consisting            of SEQ ID NOs: 1 to 29.        -   b) a nucleotide sequence which is complementary to a)            and/or,        -   c) a nucleotide sequence which is a fragment of a) or b)            and/or,        -   d) a nucleotide sequence which has an identity of at least            80% to a sequence of a), b) or c) and/or,

e) a nucleotide sequence which hybridizes under stringent conditions toa sequence of a), b), c) or d),

-   -   is used for the detection of said nucleic acid molecule(s)    -   10. The method according to any of items 1 to 9, wherein the at        least one sample is a biopsy obtained from an individual.    -   11. The method according to any of items 1 to 10, wherein the at        least one sample is a ductal fluid sample.    -   12. The method according to any of items 1 to 10, wherein the at        least one sample is a blood sample.    -   13. The method according to any of items 1 to 12, wherein the        individual is predisposed to or suffering from cancer.    -   14. The method according to any of items 1 to 13, wherein the        expression level of the at least one nucleic acid molecule is        detected by in situ detection methods.    -   15. The method of any of items 1 to 14, wherein the expression        level of the at least one nucleic acid molecule is detected by a        screening method.    -   16. The method according to item 15, wherein the screening        method comprises comparing at least two samples from the same or        different individuals.    -   17. The method according to items 15 and 16, wherein the        screening method comprises an array detection method.    -   18. The method according to any of items 15 to 17, wherein the        screening method comprises a PCR detection method such as real        time RT-PCR and/or Q-PCR.    -   19. The method according to any of items 1 to 18, for detection        of the expression level of the nucleic acid molecule for        diagnostic purposes.    -   20. The method according to any of items 1 to 19, for the        detection of the expression and or level of same of the nucleic        acid molecule prior to treatment of a clinical condition in an        individual.    -   21. The method according to any of items 1 to 20, for the        detection of the expression level of the nucleic acid molecule        during or after treatment of a clinical condition in an        individual.    -   22. A nucleic acid molecule comprising:        -   a) the nucleotide sequence of SEQ ID NO: 1 or 9 or 19 or 29            and/or,        -   b) a nucleotide sequence which is complementary to a)            and/or,        -   c) a nucleotide sequence which is a fragment of a) or b)            and/or,        -   d) a nucleotide sequence which has an identity of at least            80% to a sequence of a), b) or c) and/or,        -   e) a nucleotide sequence which hybridizes under stringent            conditions to a sequence of a), b), c) or d)    -   for the production of a pharmaceutical composition.    -   23. A pharmaceutical composition, comprising at least one        nucleic acid molecule as defined in item 22.    -   24. The pharmaceutical composition of item 23, comprising at        least two nucleic acid molecules, wherein the second or        subsequent nucleic acid molecules comprise:        -   a) the nucleotide sequence of SEQ ID NO: 2 to 8 or 1, 3, 4,            10 to 18 and 20 to 28 and/or,        -   b) a nucleotide sequence which is complementary to a)            and/or,        -   c) a nucleotide sequence which is a fragment of a) or b)            and/or,        -   d) a nucleotide sequence which has an identity of at least            80% to a sequence of a), b) or c) and/or,        -   e) a nucleotide sequence which hybridizes under stringent            conditions to a sequence of a), b), c) or d).    -   25. The pharmaceutical composition according to item 23 or 24,        for prophylactic or therapeutic applications.    -   26. The pharmaceutical composition according to any of items 23        to 25, wherein the pharmaceutical composition comprises a        pharmaceutically acceptable carrier.    -   27. The pharmaceutical composition according to any of items 23        to 26, comprising a second active ingredient.    -   28. The pharmaceutical composition according to item 27, wherein        the second active ingredient is selected from the group of        chemotherapeutics (cytostatics, anti-virals etc).    -   29. A kit comprising at least one nucleic acid molecule wherein        the nucleic acid molecule comprises:        -   a) the nucleotide sequence of SEQ ID NO: 1 or 9 or 19 or 29            and/or,        -   b) a nucleotide sequence which is complementary to a)            and/or,        -   c) a nucleotide sequence which is a fragment of a) or b)            and/or,        -   d) a nucleotide sequence which has an identity of at least            80% to a sequence of a), b) or c) and/or,        -   e) a nucleotide sequence which hybridizes under stringent            conditions to a sequence of a), b), c) or d)    -   30. The kit according to item 29 comprising at least one        additional nucleic acid molecule wherein the at least one        additional nucleic acid molecule comprises:        -   a) the nucleotide sequence of SEQ ID NO: 2 to 8 or 1, 3, 4,            10 to 18 and 20 to 28 and/or,        -   b) a nucleotide sequence which is complementary to a)            and/or,        -   c) a nucleotide sequence which is a fragment of a) or b)            and/or,        -   d) a nucleotide sequence which has an identity of at least            80% to a sequence of a), b) or c) and/or,        -   e) a nucleotide sequence which hybridizes under stringent            conditions to a sequence of a), b), c) or d).    -   31. The kit according to item 29 or 30, wherein the kit        comprises a detection device such as a microarray.    -   32. A nucleic acid construct encoding at least one nucleic acid        molecule according to any of items 1, 22 or 24.    -   33. The nucleic acid construct of item 31 encoding a promoter        enabling expression of the at least one nucleic acid molecule.    -   34. The nucleic acid construct of item 33, wherein the promoter        is selected from the group of tissue-specific promoters,        cell-type specific promoters, constitutive promoters or        inducible promoters.    -   35. A delivery vehicle comprising the nucleic acid molecule        according to any of items 1, 22 or 24 or the nucleic acid        construct of any of items 32 to 34.    -   36. A cell comprising at least one nucleic acid molecule of item        1, 22 or 24.    -   37. The cell defined in item 36, comprising the nucleic acid        construct or vehicle of any of items 32 to 36.    -   38. A pharmaceutical composition comprising any of the nucleic        acid constructs, cells or delivery vehicles of items 32 to 37.    -   39. A method of modulating the expression of a pre-microRNA or        microRNA in a cell, tissue or animal comprising contacting the        cell, tissue or animal with a composition according to any of        items 22 to 28 or 38.    -   40. A method of treating or preventing a disease or disorder        associated with aberrant expression of a pre-microRNA, microRNA        or the target of a miRNA in a cell, tissue or animal comprising        contacting the cell, tissue or animal with a composition        according to items any of the items 22 to 28 or 38.    -   41. The method according to item 40, wherein the disease or        disorder is a cancer.    -   42. The method according to item 41, wherein the cancer is a        breast cancer.    -   43. The method according to item 42, wherein the breast cancer        is selected from the group of: Luminal A, luminal B,        HER-2-overexpressing, basal and normal-like breast cancers.    -   44. The method according to any of items 39 to 43, wherein the        pre-microRNA or microRNA is selected from among any human miRNA.    -   45. The method according to any of items 39 to 43, wherein the        pre-microRNA or microRNA is selected from the group of: SEQ ID        NO:1 to 29.    -   46. The method according to any of items 39 to 45, wherein the        modulation, prophylaxis or treatment comprises administering the        pharmaceutical composition of any of items 22 to 28 or 38,    -   47. Use of a nucleic acid molecule according to any of items 1,        2, 22 or 24, for the production of a pharmaceutical composition        for the diagnosis or treatment of cancer.    -   48. The use according to item 47, wherein the cancer is a breast        cancer.    -   49. The use according to item 48, wherein the breast cancer is        from the group of: Luminal A, luminal B, HER-2-overexpressing,        basal and normal-like breast cancers.    -   50. The use according to item 47, wherein the cancer is a lung        cancer.    -   51. The use according to item 50, wherein the lung cancer is        from the group of: small cell lung cancer (SCLC) or non-small        cell lung cancer (NSCLC).

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1: Northern blots used for quantification of data presented inTable IV (upper panel) and Table V (lower panel). 5 μg of total RNA wereelectrophoretically resolved through a 12% urea-polyacrylamide gel,transferred to a nylon membrane and hybridized withradioactively-labelled StarFire probes (IDT) following vendor'srecommendations. Northern blots of selected miRNAs in the same set ofpatient cases used for microarray experiments (upper panel) and in anadditional set of 15 patients (lower panel). Molecular pathology foreach patient case is indicated in the figure.FIG. 2: Detection of miRNA expression by in situ hybridization usingFITC-labelled LNA probes in FFPE sections from breast cancer patients.Archival paraffin-embedded samples were retrieved and examined bysurgical pathologist Dr. Wendy Wells at Dartmouth Hitchcock MedicalCenter. Samples were serially sectioned at 5 μm and mounted inpositively-charged slides using floatation technique. Each slidecontains two section representing normal and tumour samples from thesame patient. One serial section was stained with hematoxylin (A-D, I-L,O-R) and another section from the same patient was used for detection ofmiRNAs by in situ hybridization. Two fields of the same section aredisplayed for each N and T samples. A-H) PS6 case is ER-PR-HER2+: miR-21is detected high levels in the periphery of tumor mass (arrows on C-Dand G-H point to the same structures); signal in N section does not comefrom epithelia (arrows on A and E), it is rather autofluorescenceemanating from stroma and adipose tissue (arrows on B and F). I-P) PS2case is ER+PR+HER2+: miR-145 is detected almost exclusively in N lobularand ductal epithelial structures (arrows on I-J and M-N), but notstroma; miR-145 expression is significantly downregulated in T section.Q-X) PS1 case is ER+PR+HER2−: miR-451 expression pattern anddistribution in epithelial structures is very similar to miR-145 (arrowson O-P and S-T).FIG. 3: Detection of miRNA expression by in situ hybridization usingFITC-labeled LNA probes in FFPE sections from a breast cancer patientcase showing a progression series from normal to malignant tissue.Specimen was prepared as described in FIG. 2. Top row panels showmiR-145 expression in indicated tissue. Middle row panels display nucleiby DAPI staining of the same section specimens used for detection ofmiR-145 expression. Low row panels display hematoxylin and eosinstaining of a different serial section used for detection of miR-145expression. Inset of top left panel indicated that miR-145 is almostexclusively expressed in basal epithelial or myoepithelials cells, andnot in luminal epithelial cells or surrounding stroma. miR-145expression is detected in fewer cells and at lower levels as the cellsbecome malignant. Tissue autoflouresence is indicated in red, andinclude signal emanating from erythrocytes and other cell types.FIG. 4: Detection of miRNA expression by in situ hybridization usingFITC-labeled LNA probes in 0.6 mm cores assembled in a tissue microarrayfrom FFPE blocks of 59 breast cancer patients. Images of fiverepresentative cases, used for data analysis in table VII, are shown.Upper panels display normal lobular and/or ductal epithelial structures.Mid and lower panel display two cores of invasive carcinoma from thesame patient as the normal tissue above. miR-145 is clearly detected innormal epithelial structures in all five patient cases, and very faintlyand/or sparsely in the invasive carcinoma tissue.FIG. 5: Cluster analysis of miRNA expression in mouse lung specimens.Colour scale represents normalized units. For the expression of eachmiRNA, the highest value was arbitrarily set at 100 and the other valueswere set accordingly. The profiles display prominently repressed (black)or induced (medium grey) miRNAs in transgenic cyclin E adenocarcinomasfrom representative wild-type [Tg+(1)] and degradation resistant cyclinE [Tg+(2)] mice. Comparisons are made to expression profiles in adjacentnormal lung. The two most repressed miRNAs in tumors were miR-34c andmiR-142-5p. The two most abundantly expressed miRNAs were miR-136 andmiR-376a. These are each novel species, not previously reported in humanlung cancers. Results were confirmed in replicate arrays, each performedat least twice in three pairs of normal-malignant lung tissues from eachline. Prior work in lung tumors recognised miR-199b, miR-126, miR-126*,miR-21, miR-146b and miR205 as being regulated. The novel miRNAsdescribed here are detailed in Table A.FIG. 6: Semi-quantitative RT-PCR assays for a representative miRNA(mi34C) differentially repressed in malignant versus normal lung from(A) transgenic mouse lines (first two pairs wild-type and seconddegradation-resistant) and from (B) paired human normal-malignant lungtissues (BAC, bronchioalveolar cancer, SC, squamous cell cancer, LClarge cell cancer, and AC, adenocarcinoma). Similar profiles are foundin these murine and human tissues, independently confirming the resultsobtained from gene profiling experiments and establishing the usefulnessof these transgenic lines to predict miRNA expression patterns in humantissues.FIG. 7: Detection of miRNA expression by in situ hybridization usingFITC-labeled LNA probes in FFPE sections of murine model of lung cancer.Transgenic animals overexpressing human cyclin E were sacrificed. Lungtissue was dissected and fixed with 10% buffered formalin for at least12 hrs. Then, samples were embedded in paraffin, serially sectioned at 5μm and mounted in positively-charged slides using floatation technique.Each slide represents adjacent normal lung tissue and human cyclinE-induced displasia and adenocarcinoma. miR-145 is expressed abundantlyin normal lung structures, but it is detected at low levels orcompletely abscent in adenocarcinoma lesion.FIG. 8: The most prominently over-expressed miRNAs in murine transgeniclung cancers relative to adjacent normal lung tissues. A) MiRNAprofiling of lung adenocarcinomas and adjacent normal lung tissues frommurine wild-type (line 2) and degradation-resistant (line 4) cyclin Etransgenic lines. MiR-136, miR-376a, miR-31, miR-205, miR-337, miR-410,miR-379, miR-127, miR-431 and miR-21 were each significantlyover-expressed in lung adenocarcinomas versus normal lung. B)Quantification is displayed of the over-expressed miRNAs in murine lungcancers versus normal lung tissues in panel A.FIG. 9: ISH assays for representative over-expressed miRNAs in lungcancers. These assays were conducted on adenocarcinomas and adjacentnormal lung tissues from cyclin E transgenic mice and from human pairednormal-malignant lung tissues. Over-expression of miR-21 and miR-31 wasdetected in malignant versus normal lung tissues. The 18S rRNA signalserved as a positive control for integrity of RNA. Hematoxylin and eosin(H&E) staining of the indicated tissue sections is shown.FIG. 10: Validation of miR-136, miR-376a and miR-31 expression profilesby real-time RT-PCR assays performed on RNA isolated from the indicatedmurine cyclin E transgenic lines and tissues from paired humannormal-malignant lung tissues. A) Real-time RT-PCR assays for miR-136,miR-376a and miR-31 were performed with symbols depicting T, malignanttumor; N, adjacent normal murine lung; and Tg−, murine non-transgenicFVB normal lung tissue. Results were normalized to expression levelsdetected in FVB murine Tg− lung tissues. B) Real-time RT-PCR assays formiR-136, miR-376a and miR-31 were independently performed on pairedhuman normal-malignant lung tissues, with symbols depicting T, malignanttumor; N, adjacent normal human lung; AD, adenocarcinoma; LC, large cellcarcinoma; BAC, bronchioalveolar carcinoma; and SC, squamous cellcarcinoma. Results were normalized to expression levels measured innormal human lung. Standard deviation bars are displayed.FIG. 11: Validation of miR-31 expression by real-time RT-PCR assaysperformed on the indicated cell lines as well as malignant and adjacentnormal lung tissues from murine cyclin E transgenic lines andindependently on paired normal-malignant human lung tissues. A)Real-time RT-PCR assays for miR-31 in murine ED-1, ED-2, C10 cells andmurine cyclin E transgenic malignant versus normal lung tissues withsymbols depicting T, malignant transgenic lung tumor; N, adjacent normalmurine transgenic lung; and Tg−, murine non-transgenic FVB normal lungtissue. Results represented the fold changes of miR-31 expressionrelative to sno-135 (control small RNA). B) Real-time RT-PCR assays formiR-31 were performed on RNA derived independently from BEAS-2B, HOP62,H226, H522 and A549 cells as well as from paired human normal-malignantlung tissues, with symbols depicting T, malignant lung tumor; N,adjacent normal lung; AD, adenocarcinoma; LC, large cell carcinoma; BAC,bronchioalveolar carcinoma; and SC, squamous cell carcinoma. Resultsrepresented the fold of miR-31 relative to RNU6B (control small RNA).FIG. 12: Regulation of miR-31 expression affects lung cancer cellproliferation. A) Proliferation of ED-1 and ED-2 murine lung cancercells and H23 and H226 human lung cancer cells was each suppressed byengineered miR-31 knock-down by anti-miR-31 transfection, but murine C10pulmonary epithelial cell proliferation was less significantlysuppressed and BEAS-2B human immortalized bronchial epithelial cellgrowth was not significantly suppressed by this transfection. Thesymbols are: *, P<0.05; **, P<0.01; ***, P<0.0001. The lower paneldisplays real-time RT-PCR assays that confirm miR-31 repression and allP values were <0.0001. B) Significant repression of ED-1 and ED-2 cellgrowth caused by anti-miR-31 was antagonized by transfection ofpre-miR-31 at 48 hours after anti-miR-31 transfection. The left panelfor each lung cancer cell line displays proliferation and the rightpanel presents the real-time RT-PCR assay results confirming theexpected miR-31 expression levels of these transfectants. In all groupsP values were <0.0001.FIG. 13: Repression of miR-31 expression significantly affects murinelung cancer clonal growth and tumorigenicity. A) Colony formation assaysfor ED-1 and ED-2 murine lung cancer cells were independently suppressedrelative to controls (ctrl) by engineered knock-down of miR-31 throughtransient anti-miR-31 transfection. The stained colonies are displayedin the lower panel. B) Repression of in vivo lung tumorigenicity in theindicated transfected ED-1 cells following FVB mouse tail-veininjections. The bars represent lesion numbers in the lungs. Forty micein total were used and each group had 10 mice with results pooled fromtwo independent experiments, as described in the Methods and Materials.The symbols displayed are: *, P<0.05; ** and P<0.01.FIG. 14: LATS2 and PPP2R2A are miR-31 target mRNAs. Real-time RT-PCRassays confirmed that the A) LATS2 and B) PPP2R2A expression levels wereeach down-regulated by pre-miR-31 and up-regulated by anti-miR-31transfections independently performed in ED-1, ED-2, C-10, H23, H226 andBEAS-2B cells. All transfectant groups had P values <0.0001.FIG. 15: The proliferation of murine lung cancer cells followingregulated expression of miR-31, LATS2 or PPP2R2A. The panels display A)ED-1 cells and C) ED-2 cells and their growth was suppressed bytransfection of anti-miR-31. This was antagonized by either LATS2 orPPP2R2A targeting siRNA transfections performed 48 hours afteranti-miR-31 transfection. Two independent siRNAs were each used totarget LATS2 as well as PPP2R2A. The symbols displayed are: **, P<0.01and ***, P<0.0001. The mRNA levels of LATS2 and PPP2R2A are presentedfor the indicated transfectants in panels B) ED-1 cells and D) ED-2cells following real-time RT-PCR assays. All results were normalized tothe combined anti-miR-neg and the negative control siRNA transfectantgroup. In all groups P values were <0.0001.FIG. 16: Validation of LATS2 and PPP2R2A expression profiles byreal-time RT-PCR assays performed on RNA isolated from the indicatedmurine cyclin E transgenic lines and tissues from paired humannormal-malignant lung tissues. Real-time RT-PCR assays for: A) LATS2 andB) PPP2R2A were performed with symbols depicting T, malignant lungtumor; N, adjacent normal murine lung; and Tg−, murine non-transgenicFVB normal lung. Results were normalized to expression withinnon-transgenic FVB mouse lung tissues. Real-time RT-PCR assays for C)LATS2 and D) PPP2R2A were independently performed on paired humannormal-malignant lung tissues, with symbols depicting T, malignant lungtumor; N, adjacent normal lung tissues; AD, adenocarcinoma; LC, largecell carcinoma; BAC, bronchioalveolar carcinoma; and SC, squamous cellcarcinoma. Results were normalized to expression in normal human lungtissues. Standard deviation bars are displayed. In all groups P valueswere <0.001.

EXAMPLES Example 1

Identification of Novel, Differentially Expressed miRNAs in BreastTumoursTable I. The 10 Breast Cancer Cases Used to Identify Novel,Differentially Expressed miRNAs in Example 1.

Four of these cases, all which are ER+(i.e. BC#2, 4, 6, 8), have amatching normal tissue section. Most of these tumours are infiltratingductal carcinomas, of high grade. These tumours represent a range ofstages due to tumour size from 1.8 to 7.2 cm and node status.

Brief name Subtype Comments Type Grade Size Nodes BC#1 ER+/PR+/HER2−Normal N/A N/A N/A N/A BC#2 ER+/PR+/HER2− ER+ Tumor IDCa LG 1.8 cm 1 of4 BC#3 ER+/PR+/HER2− Normal N/A N/A N/A N/A BC#4 ER+/PR+/HER2− ER+ TumorIDCa HG 2.0 cm 1 of 1 BC#5 ER+/PR+/HER2− Normal N/A N/A N/A N/A BC#6ER+/PR+/HER2− ER+ Tumor IDCa HG 7.0 cm 3 of 7 BC#7 ER+/PR+/HER2− NormalN/A N/A N/A N/A BC#8 ER+/PR+/HER2− ER+ Tumor IDCa HG 4.5 cm Not doneBC#9 ER−/PR−/HER2+ ER− Tumor IDCa HG 2.1 cm 1 of 1 BC#10 ER−/PR−/HER2−ER− Tumor Met Ca HG 2.9 cm 0 of 4 BC#11 ER−/PR−/HER2− ER− Tumor IDCa HG2.8 cm 0 of 7 BC#12 ER−/PR−/HER2+ ER− Tumor IDCa HG 3.3 cm 19 of 22BC#13 ER−/PR−/HER2− ER− Tumor IDCa HG 2.9 cm 5 of 8 BC#14 ER−/PR−/HER2−ER− Tumor IDCa HG 7.2 cm 17 of 20 Key for Table I definitions: IDCa =infiltrating ductal carcinoma Met Ca = metaplastic carcinoma HG = highgrade IG = intermedoate grade LG = low grade

A) Isolation of the Breast Tumour Samples

Tumour samples were obtained through the research pathology services atDartmouth-Hitchcock Medical Center. Tumour samples were excised frompatients as result of surgery (lumpectomies or mastectomies). Tumoursamples from the operation room were delivered to surgical pathologylaboratory for routine diagnosis and archive of the samples.

B) Total RNA Extraction

All breast tumour samples described in detail in Table I, were obtainedas frozen specimens preserved in liquid nitrogen. Total RNA wasextracted using Trizol reagent according to the manufacturer'sinstructions (Invitrogen).

C) RNA Labelling for miRNA Microarray Profiling

Total RNA extracted from the breast tumours (Table I) was 3″end labeledusing T4 RNA ligase and Cy3- or Cy5-labeled RNA linker(5″-PO₄-rUrUrU-Cy3/dT-3″ or 5″-PO₄-rUrUrU-Cy5/dT-3′). The labelingreactions contained 3.5 μg total RNA, 15 μM RNA linker, 50 mM Tris-HCl(pH 7.8), 10 mM MgCl₂, 10 mM DTT, 1 mM ATP, 16% polyethylene glycol and5 unit T4 RNA ligase (Ambion, USA) and were incubated at 30° C. for 2hours followed by heat inactivation of the T4 RNA ligase at 80° C. for 5minutes.

D) Microarray Hybridization and Post-Hybridization Washes

LNA-modified oligonucleotide capture probes comprising probes for allannotated miRNAs annotated from mouse (Mus musculus) and human (Homosapiens) in the miRBase MiRNA database Release 7.1 including a set ofpositive and negative control probes were purchased from Exiqon, Denmarkand used to print the LNA microarray platform. The capture probescontain a 5″-terminal C6-amino modified linker and were designed to havea T_(m) of 72° C. against complementary target miRNAs by adjustment ofthe LNA content and length of the capture probes. The capture probeswere diluted to a final concentration of 10 μM in 150 mM sodiumphosphate buffer (pH 8.5) and spotted in quadruplicate onto Codelinkslides (Amersham Biosciences) using the MicroGrid II arrayer fromBioRobotics at 45% humidity and at room temperature. Spotted slides werepost-processed as recommended by the manufacturer.

Labelled RNA was hybridized to the LNA microarrays overnight at 65° C.in a hybridization mixture containing 4×SSC, 0.1% SDS, 1 μg/μl HerringSperm DNA and 38% formamide. The hybridized slides were washed threetimes in 2×SSC, 0.025% SDS at 65° C., followed by three times in0.08×SSC and finally three times in 0.4×SSC at room temperature.

E) Array Scanning, Image Analysis and Data Processing

The microarrays were scanned using the ArrayWorx scanner (AppliedPrecision, USA) according to the manufacturer's recommendations. Thescanned images were imported into TIGR Spotfinder version 3.1 (Saeed etal., 2003) for the extraction of mean spot intensities and median localbackground intensities, excluding spots with intensities below medianlocal background+4× standard deviations. Background-correlatedintensities were normalized using variance stabilizing normalizationpackage version 1.8.0 (Huber et al., 2002) for R (www.r-project.org).Intensities of replicate spots were averaged using Microsoft Excel.Probes displaying a coefficient of variance >100% were excluded fromfurther data analysis.

Table II. Differential Expression of Nine Different miRNAs in BreastTumour Samples as Detected by LNA Microarray Expression Profiling.

The displayed expression levels show the average intensities from two orthree replica experiments.

TABLE II miRNA Normal tissue ER+tumors ER−tumors name BC#1 BC#3 BC#5BC#7 BC#2 BC#4 BC#6 BC#8 BC#9 BC#10 BC#11 BC#12 BC#13 BC#14 miR-451 36246578 8308 1383 6051 2155 1132 193 150 452 857 1139 428 2166 miR-143 11481947 1064 5161 1368 1177 812 1448 380 682 655 752 800 1108 miR-145 20733054 702 4647 1290 1061 401 1280 245 392 348 368 504 598 miR-21 19824776 4921 7810 13785 10311 5640 7095 2853 12869 12142 14472 8903 14917miR-141 506 423 845 386 318 796 2035 780 1224 752 2362 692 516 1255miR-200b 968 1596 1777 1047 1145 2340 2283 1675 3614 1540 2393 2089 15031878 miR-200c 941 1232 1972 1410 577 1758 3324 1948 3840 1602 4814 22622362 2833 miR-221 803 920 1098 2057 1235 871 1013 1286 923 1294 19121141 913 1488 miR-222 656 644 778 1645 805 624 734 1252 677 1068 1793904 845 1263Table III. Log 2-Transformed Expression Ratios of Nine microRNAs inBreast Tumour Samples the Indicated Samples.

The ER+ tumours (BC#2, 4, 6, 8) are paired with their matching normaltissue section, while BC#9-14 are paired with BC#N. BC #N represents theaverage expression of the given miRNAs in the normal tissue samples BC#1, 3, 5, 7. Positive log 2-ratios indicate upregulation, and negativevalues indicate downregulation of the given miRNA.

TABLE III ER+tumors ER−tumors miRNA name BC#2/#1 BC#4/#3 BC#6/#5 BC#8/#7BC#9/#N BC#10/#N BC#11/#N BC#12/#N BC#13/#N BC#14/#N miR-451 0.74 −1.61−2.88 −2.84 −5.05 −3.46 −2.54 −2.13 −3.54 −1.2 miR-143 0.25 −0.73 −0.39−1.83 −2.61 −1.77 −1.83 −1.63 −1.54 −1.07 miR-145 −0.68 −1.53 −0.81−1.86 −3.42 −2.74 −2.91 −2.83 −2.38 −2.13 miR-21 2.8 1.11 0.2 −0.14−0.77 1.4 1.32 1.57 0.87 1.61 miR-141 −0.67 0.91 1.27 1.01 1.18 0.482.13 0.36 −0.07 1.22 miR-200b 0.24 0.55 0.36 0.68 1.42 0.19 0.83 0.630.16 0.48 miR-200c −0.71 0.51 0.75 0.47 1.47 0.21 1.79 0.7 0.77 1.03miR-221 0.62 −0.08 −0.12 −0.68 −0.4 0.09 0.65 −0.1 −0.42 0.29 miR-2220.3 −0.04 −0.08 −0.39 −0.46 0.2 0.95 −0.04 −0.14 0.44

F) Northern Blot Analysis

Total RNA of each breast tissue sample (Table I), 5 μg per lane, informamide loading buffer (Ambion) was heated at 90° C. for 3 min, andelectrophoretically separated through a 12% denaturingurea-polyacrylamide gel at 125 V for 1.5 h in 1×TBE at 25° C. RNA waselectrophoretically transferred to a Genescreen plus (NEN) at 80 V for 1h in 0.5×TBE at 4° C. RNA was cross-linked to the membrane by UVirradiation (1200 mJ; Stratagene UV Stratalinker), subsequently themembrane was baked at 80° C. for 30 min. miRNA and snoRNA U6 antisenseStarFire (Integrated DNA Technologies) radiolabeled probes were preparedby incorporation of [alpha-³²P]dATP 6000 Ci/mmol as recommended by thevendor.

The StarFire probe sequences are listed below. For miRNA probes,membranes were hybridized for 24 h at 42° C. in 7% SDS. 0.2MNa2PO4, pH7.2, and washed twice with 2×SSPE 0.1% SDS, and once with 1×SSPE 0.1%SDS, and 0.5×SSPE 0.1% SDS at 42° C. For U6 probe, membranes werehybridized overnight at 42° C. in 7% SDS 0.05 M Na2PO4, pH 7.2, andwashed twice with 1×SSPE 0.1% SDS, and once with 0.5×SSPE 0.1% SDS and0.1×SSPE 0.1% SDS at 42° C.

Table IV. Confirmation of Differentially Expressed miRNAs by NorthernAnalysis in Breast Tumour Samples.

Expression of listed miRNAs was determined by Northern analysis in thesame breast tumour samples: BC#1-14 (Table I and II) used for LNAmicroarray profiling. Radioactive signal of miRNA bands was quantifiedusing ImageQuant and corrected using U6 as loading control. Relativeintensities of miRNA expression levels are displayed (− no expression, +low signal to +++ highest signal). We observed a high concordancebetween microarray and Northern results.

In table IV, miR-451, -21, 141, 200b, -221, and -222 correspond to SEQID NOs 30, 31, 32, 33, 34 and 35, respectively.

TABLE IV miRNA Probe Normal tissue ER+tumors ER−tumors name sequenceBC#1 BC#3 BC#5 BC#7 BC#2 BC#4 BC#6 BC#8 BC#9 BC#10 BC#11 BC#12 BC#13BC#14 miR-451 AAACTCAGTA + ++ +++ ++ ++ − − − − + − − + + ATGGTAACGGTTTmiR-21 TCAACATCAG − + + + ++ − − ++ +++ +++ +++ ++ +++ +++ TCTGATAAGCTAmiR-141 CCATCTTTAC − − − − − − − − ++ ++ ++ + ++ +++ CAGACAGTGTTmiR-200b GTCATCATTA − − + − − − − + +++ +++ ++ + + + CCAGGCAGTATTAmiR-221 GAAACCCAGC − − − + + − − + − +++ + − + + AGACAATGTAGCT miR-222TCGATGTAGA − − − − − − − − + + + + + + CCGATGACCCAGAGTable V. Expression Profiling of miRNAs by Northern Analysis in anAdditional Set of 11 Normal/Tumour Matched Samples.

Tumour samples 5, 6, 7, 8 (matched to normal 1, 2, 3, 4, respectively)and unmatched 9 are ER+PR+HER2− tumours; tumour samples 14, 15, 16(matched to normal 10, 11, 12, 13, respectively) are ER+PR+HER2+tumours; tumour samples 20, 21 (matched to normal 18, 19,respectively—though no significant amount of RNA was obtained from thesenormal samples) and unmatched 22 are ER-PR-HER2+ tumours; tumour samples24 (matched to are normal 23) and unmatched 25 and 26 ER-PR-HER2−tumours (no significant amount of RNA was obtained). Experiments wereconducted as described for Table IV. Samples 18, 19, 25 and 26 werediscarded from data analysis due to RNA underloading and thus, are notincluded in this table.

In table V, miR-143, -145, -451, -21, -141, and -221 correspond to SEQID NOs 36, 37, 30, 31, 32 and 34, respectively.

TABLE V miRNA Probe Normal tissue ER+TUMORS name sequence 1 2 3 4 10 1112 13 18 19 23 5 6 7 8 9 miR- TGAGCTA + + ++ + +++ + + − * * ++ + − + +− 143 CAGTGCT TCATCTC A miR- AAGGGAT ++ ++ ++ ++ ++ +++ ++ − * *++ + + + − − 145 TCCTGGG AAAACTG GAC miR- AAACTCA − − − − +++ − −− * * + − − − − − 451 GTAATGG TAACGGT TT miR-21 TCAACAT − − − − ++ − −− * * + + + + ++ − CAGTCTG ATAAGCT A miR- CCATCTT + − + + + + − − * * +++ − ++ +++ + 141 TACCAGA CAGTGTT miR- GAAACCC + − +++ − + + + − * * + +− ++ + − 221 AGCAGAC AATGTAG CT miRNA Probe ER+TUMORS ER−TUMORS namesequence 14 15 16 17 20 21 22 24 25 26 miR- TGAGCTA +++ + + + + + + +− + 143 CAGTGCT TCATCTC A miR- AAGGGAT + + + + + + + − − − 145 TCCTGGGAAAACTG GAC miR- AAACTCA + − − + + − − − − − 451 GTAATGG TAACGGT TTmiR-21 TCAACAT ++ ++ − ++ +++ + + ++ + + CAGTCTG ATAAGCT A miR- CCATCTT+++ +++ + + ++ + + + − + 141 TACCAGA CAGTGTT miR- GAAACCC ++ + + + −++ + + − − 221 AGCAGAC AATGTAG CT

G) Results

Analysis of the miRNA microarray data revealed that miR-451 wassignificantly down-regulated in all high grade ER-positive breasttumours as well as in all ER-negative tumours, compared to the normalbreast samples. In addition, both miR-143 and miR-145 were founddown-regulated in all breast tumour samples studied in this example,while miR-21 and miR-141 were up-regulated in most of the tumoursamples. miR-200b and miR-200c showed upregulation in some of the tumoursamples. Northern analysis of examined miRNAs corroborates thesefindings.

Example 2

Determination of Spatial Distribution of Selected miRNAs inFormalin-Fixed Paraffin-Embedded Tumour Sections by LNA In SituHybridization.Table VI. In Situ Detection of Four miRNAs in Tumour Sections of 9 Casesof Breast Cancer.

All these FFPE tumour sections were accompanied with a matching normalsection from the same patient. This table summarizes results of theexperiments looking at 5-10 random of each normal and tumour sectionfields under the epifluorescence microscope. Signal intensity wasvisually quantified from no expression (0) to high expression (3) instroma and epithelial structures (lobules and milk ducts) for normalsection and only in tumour mass for tumour section.

TABLE VI Pa- miR-21 tient Tumor Tu- miR-141 miR-145 miR-451 Subtype CaseGrade Stroma EpiStruct mor Stroma EpiStruct Tumor Stroma EpiStruct TumorStroma EpiStruct Tumor ER+/PR+/ PS1 IG 0 2 0 N/A N/A N/A 0 2 0 0 3 0HER2− ER+/PR+/ PS2 HG 0 1 0 N/A N/A N/A 0 3 1 1 2 0 HER2− ER+/PR+/ PS3HG N/A N/A N/A 0 0 0 1 3 1 0 1 0 HER2− ER+/PR+/ PS4 HG 1 0 2 0 1 0 0 2 00 1 0 HER2+ ER+/PR+/ PS5 HG 0 1 2 N/A N/A N/A 0 3 0 0 2 0 HER2+ ER−/PR−/PS6 HG 0 1 3 1 1 1 0 2 1 0 1 1 HER2+ ER−/PR−/ PS7 HG 0 0 0 0 1 0 0 3 0N/A N/A N/A HER2+ ER−/PR−/ PS8 HG N/A N/A N/A N/A N/A N/A 0 2 0 N/A N/AN/A HER2− ER−/PR−/ PS9 HG 0 0 0 1 1 2 0 2 0 0 1 1 HER2− Definitions IG =intermedoate grade HG = high grade EpiStruct = Epithelial structures(ducts and lobules)

5′ FITC-labeled LNA probes complementary for hsa-miR-21, hsa-miR-141,hsa-miR-145 and hsa-miR451 were purchased from Exiqon, Denmark and usedin this in situ hybridization experiments. In situs with miR-200 andmiR-222 required optimization.

Table VII. In Situ Detection of miR-145 in 0.6 Mm Core of a TissueMicroarray Representing Normal, In Situ Carcinoma and Invasive CarcinomaTissue from 59 Breast Cancer Patients.

This table summarizes of tissue microarray experiment for miR-145expression. Tissue microarrays were hybridized in two independentexperiments with LNA probes against miR-145. Signal intensity wasvisually quantified from no expression (0) to high expression andcontinuous pattern of expression (3). No signal is represented with 0,low expression/expressing cells are sparsely distributed and representedwith +, intermediate expression/discontinuous or intermittent pattern ofexpression is represented with ++, high expression/continuous pattern isrepresented with +++. Cores that did not represent the appropriatetissue and or had insufficient material to diagnose are indicated with*. Cases are sorted descendingly by signal in normal tissue.

TABLE VII miR-145 3 16 18 43 49 35 1 5 28 54 31 30 39 51 37 58 33 7Normal +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ ++ ++ ++ ++ ++ ++ ++Carcinoma * * * * 0 0 0 + ++ ++ * * 0 0 0 0 0 + in situ Invasive 0 0 0 00 0 * 0 0 * 0 0 0 0 + ++ * 0 Carcinoma Invasive 0 0 0 0 0 0 0 0 0 * 00 * 0 * 0 * 0 Carcinoma miR-145 25 2 6 34 48 15 46 27 41 44 45 14 26 1112 17 55 56 20 29 9 19 22 Normal + + + + + + + + + + + 0 0 0 00 * * * * * * * Carcinoma * * * * 0 0 0 0 0 + + 0 0 0 0 0 * * * * * * *in situ Invasive 0 0 0 * * 0 0 0 0 0 0 0 0 0 0 0 * * 0 0 0 0 0 CarcinomaInvasive 0 0 0 * 0 0 * 0 0 0 0 0 0 0 * 0 * 0 0 0 0 0 0 Carcinoma miR-14523 36 50 52 32 40 42 13 4 8 24 38 47 53 10 21 57Normal * * * * * * * * * * * * * * * * * Carcinoma * * * 0 0 0 00 + + + + + 0 * * 0 in situ Invasive 0 0 0 * 0 0 0 * 0 0 0 0 0 0 * 0 *Carcinoma Invasive * 0 0 0 0 * 0 0 0 0 0 0 * 0 * 0 * Carcinoma

A) Preparation of the Formalin-Fixed, Paraffin-Embedded Sections for InSitu Hybridization

Archival paraffin-embedded samples were retrieved and examined bysurgical pathologist Dr. Wendy Wells at Dartmouth Hitchcock MedicalCenter. Samples were serially sectioned at 5 μm and mounted inpositively-charged slides using floatation technique. Each slidecontains two sections representing normal and tumour samples from thesame patient or 0.6 mm cores from tissue microarray blocks. Slides werestored at 4° C. until the experiments were conducted. The molecular andclinical history of these samples is available to us. These samplesrepresent breast cancer subtypes (ER+vs ER− and HER2+ vs HER2−).

B) In Situ Hybridization

Sections on slides were deparaffinized in xylene and then rehydratedthrough an ethanol dilution series (from 100% to 25%). Slides weresubmerged in DEPC-treated water and subject to HCl and 0.2% Glycinetreatment, re-fixed in 4% paraformaldehyde and treated with aceticanhydride/triethanolamine; slides were rinse in several washes of 1×PBSin-between treatments. Slides were pre-hybridized in hyb solution (50%formamide, 5×SSC, 500 mg/mL yeast tRNA, 1×Denhardt) at 50° C. for 30min. Then, 3 pmol of the FITC-labeled LNA probe complementary to eachselected miRNA was added to the hyb. solution and hybridized for one hrat a temperature 20-25° C. below the predicted T_(m) of the probe(typically between 45-55° C. depending on the miRNA). After washes in0.1× and 0.5×SCC at 65° C., a tyramide signal amplification reaction wascarried out using the Genpoint Fluorescein (FITC) kit (DakoCytomation,Denmark) following the vendor's recommendations. Finally, slides weremounted with Prolong Gold solution.

Fluorescence reaction was allowed to develop for 16-24 hr beforedocumenting expression of the selected miRNA with epifluorescencemicroscope.

C) Results

The results show that miR-145 and miR-451 were specifically expressedwithin epithelial structures of normal tissue, whereas no expression orlow expression (in HER2+ tumours) was detected in tumour sections withthe exception of normal epithelial structures flanking the tumour mass.This demonstrates that miR-145 and miR-451 are expressed in theepithelial cells from which the carcinoma arises and thus the absence ofthese miRNAs in the tumour mass strongly suggest that they aredownregulated as a direct consequence of the evolution of tumour. Insome instances, miR-21 and miR-141 expression was upregulated in thetumour mass, whereas no signal or low signal was observed in normalstroma and/or epithelial structures. This is in agreement with theexpression data obtained by microarray profiling and Northern analysisin example 1.

Example 3

The Novel microRNA Biomarker Sequences1. The following miRNAs are downregulated in breast tumours compared tonormal breast biopsies both analyzing whole samples by Northern and LNAmicroarray technique and analyzing spatial distribution of miRNAs withinepithelial structures of breast tissue by in situ hybridizationtechnique:

(SEQ ID NO: 1) A) hsa-miR-451; 5′-aaaccguuaccauuacugaguuu-3′(SEQ ID NO: 2) B) hsa-miR-143; 5′-ugagaugaagcacuguagcuca-3′(SEQ ID NO: 3) C) hsa-miR-145; 5′-guccaguuuucccaggaaucccuu-3′2. The following miRNAs are upregulated in specific subtypes of breasttumours compared to normal breast biopsies and in some instanceadditional evidence of this fact was obtained by in situ hybridizationtechnique:

(SEQ ID NO: 4) A) hsa-miR-141; 5′-uaacacugucugguaaagaugg-3′(SEQ ID NO: 5) B) hsa-miR-200b; 5′-uaauacugccugguaaugaugac-3′(SEQ ID NO: 6) C) hsa-miR-200c; 5′-uaauacugccggguaaugaugg-3′(SEQ ID NO: 7) D) hsa-miR-221; 5′- agcuacauugucugcuggguuuc-3′(SEQ ID NO: 8) E) hsa-miR-222; 5′- agcuacaucuggcuacugggucuc-3′(SEQ ID NO: 17) F) hsa-miR-21; 5′-uagcuuaucagacugauguuga-3′Table VIII. Chromosomal Location of miRNAs as Listed in miRbase.

Notice that differentially expressed miRNAs are tightly linked in thegenome and are related in sequence with the exception of miR-143 andmiR-145 and miR-144 and miR-451. Tightly linked or clustered miRNAs arethought to be regulated by the common regulatory signals and in mostcases are co-transcribed in a long primary transcript from whichindividual miRNA hairpin precursors are processed.

miRNA Accession nr Chrom# from to hsa-mir-200b MI0000342 1 11424071142501 hsa-mir-200a MI0000737 1 1143166 1143255 hsa-mir-429 MI0001641 11144308 1144390 hsa-mir-143 MI0000459 5 148788674 148788779 hsa-mir-145MI0000461 5 148790402 148790489 hsa-mir-200c MI0000650 12 69431236943190 hsa-mir-141 MI0000457 12 6943521 6943615 hsa-mir-451 MI000172917 24212513 24212584 hsa-mir-144 MI0000460 17 24212677 24212762hsa-mir-221 MI0000298 X 45361839 45361948 hsa-mir-222 MI0000299 X45362675 45362784 hsa-mir-21 MI0000077 17 55273409 55273480

Example 4

Method for Quantification of mRNA Levels by Real-Time RT-PCR Assay.

First strand cDNA synthesis is carried out using 1 μg of a total RNA,random hexamer primers and SuperScript II reverse transcriptase.Following spin column purification the volume of the cDNA preparation isadjusted to 100 μl per 1 μg input total RNA and used as template in thereal-time PCR reaction. Quantitative real-time PCR assays for the mRNAof interest is performed using 1× TaqMan Universal PCR Master Mix, NoAmpErase UNG and 1× TaqMan Assays-on-Demand, Gene Expression Product forthe given mRNA (or 1× TaqMan Pre-Developed Assay Reagent endogenouscontrol for the β-actin mRNA (TaqMan PDAR human ACTB; Cat no. 4310881E)in an ABI 7900HT Sequence Detection System as follows: 10 mindenaturation at 95° C. followed by 40 cycles of denaturation for 15 s at95° C. and annealing and elongation for 1 min at 60° C. using 2 μL ofthe five times diluted cDNA reaction from above as template in a finalvolume of 25 μL. The relative gene expression level of the mRNA can becalculated as described in the user bulletin #2: ABI PRISM 7700 SequenceDetection System. Unless otherwise stated all reagents and equipment arepurchased from Applied Biosystems, USA.

Example 5

Methods for Real-Time Quantification of Mature miRNAs by Stem-LoopRT-PCR

Reverse Transcriptase Reaction

The reverse transcriptase reaction is carried out using 1-25 ng ofpurified total RNA, 50 nM stem-loop RT primer for a given miRNA (AppliedBiosystems, USA), 1×RT buffer (Applied Biosystems, USA), 0.25 mM each ofdNTPs, 3.33 U/μl MultiScribe reverse transcriptase (Applied Biosystems,USA) and 0.25 U/μl RNase inhibitor (Applied Biosystems, USA). The 7.5 μlreactions are incubated in an Applied Biosystems 9700 Thermocycler in a96- or 384-well plate for 30 min at 16° C., 30 min at 42° C., 5 min at85° C. and then held at 4° C. The reactions, including no-templatecontrols and RT minus controls, are run in duplicate.

Quantification by Real-Time PCR

Real-time PCR is performed using a standard TaqMan PCR kit protocol onan Applied Biosystems 7900HT Sequence Detection System (AppliedBiosystems, USA). The 10 μl PCR includes 0.67 μl RT product, 1× TaqManUniversal PCR Master Mix (Applied Biosystems, USA), 0.2 μM TaqMan probe,1.5 μM forward primer and 0.7 μM reverse primer. The reactions areincubated in a 384-well plate at 95° C. for 10 min, followed by 40cycles of 95° C. for 15 s and 60° C. for 1 min. All reactions are run intriplicate. The threshold cycle (CT) is defined as the fractional cyclenumber at which the fluorescence passes the fixed threshold. TaqMan CTvalues are converted into absolute copy numbers using a standard curvefrom synthetic lin-4 miRNA.

Example 6

List of Putative Target Genes for Human miR-451Table IX. List of Exemplary Target Genes for Human miR-451 as Predictedby the miRBase Targets Version 2.0 Program (Http COLON SLASH SLASHmicroRNA DOT sanger DOT ac DOT uk/targets/v2/).

TABLE IX No. Total Cons Gene Name Transcript id Description P valueSites Spec FTS ENST00000300245 Fused toes protein homolog (Ft1).1.51E−06 1 8 [Source: Uniprot/SWISSPROT; Acc: Q9H8T0] NP_054907.1ENST00000291386 Ssu72 RNA polymerase II CTD 1.85E−05 1 6 phosphatasehomolog [Source: RefSeq_peptide; Acc: NP_054907] ING3 ENST00000315870Inhibitor of growth protein 3 2.98E−05 3 8 (p47ING3 protein). [Source:Uniprot/SWISSPROT; Acc: Q9NXR8] NSMAF ENST00000038176 Protein FAN(Factor associated with 4.75E−05 1 6 N-SMase activation) (Factorassociated with neutral- sphingomyelinase activation). [Source:Uniprot/SWISSPROT; Acc: Q92636] PITPNB ENST00000320996Phosphatidylinositol transfer protein 5.74E−05 1 7 beta isoform (Ptdlnstransfer protein beta) (PtdlnsTP) (PI-TP-beta). [Source:Uniprot/SWISSPROT; Acc: P48739] PHTF2 ENST00000248550 putativehomeodomain transcription 6.01E−05 1 6 factor 2 [Source: RefSeq_peptide;Acc: NP_065165] PMM2 ENST00000268261 Phosphomannomutase 2 (EC 6.09E−05 15 5.4.2.8) (PMM 2). [Source: Uniprot/SWISSPROT; Acc: O15305] APXLENST00000275869 Apical-like protein (APXL protein). 8.09E−05 1 4[Source: Uniprot/SWISSPROT; Acc: Q13796] NEDD9 ENST00000265010 Enhancerof filamentation 1 (HEF1) 8.21E−05 1 9 (CRK-associated substrate-relatedprotein) (CAS-L) (CasL) (p105) (Neural precursor cell expresseddevelopmentally down-regulated 9). [Source: Uniprot/SWISSPROT; Acc:Q14511] TRAF7 ENST00000326181 E3 ubiquitin protein ligase TRAF7 9.31E−051 5 (EC 6.3.2.—) (TNF receptor- associated factor 7) (Ring finger and WDrepeat domain 1) (RING finger protein 119). [Source: Uniprot/SWISSPROT;Acc: Q6Q0C0] TMEM33 ENST00000264452 Transmembrane protein 33 (DB839.46E−05 3 4 protein). [Source: Uniprot/SWISSPROT; Acc: P57088]NP_060498.2 ENST00000314471 PREDICTED: similar to CDNA 0.000108 1 5sequence BC042901 [Source: RefSeq_peptide_predicted; Acc:XP_218384]PREDICTED: similar to CDNA sequence BC042901 [Source:RefSeq_peptide_predicted; Acc: XP_218384] BY ORTHOLOGY TO:ENSRNOT00000026839 LDHA ENST00000227157 L-lactate dehydrogenase A0.000109 1 5 chain (EC 1.1.1.27) (LDH-A) (LDH muscle subunit) (LDH-M)(Proliferation-inducing gene 19 protein). [Source: Uniprot/SWISSPROT;Acc: P00338] MORC4_HUMAN ENST00000255495 MORC family CW-type zinc0.000118 1 4 finger 4 (Zinc finger CW-type coiled-coil domain protein2). [Source: Uniprot/SWISSPROT; Acc: Q8TE76] TRIM66 ENST00000299550Tripartite motif protein 66. 0.000147 1 4 [Source: Uniprot/SWISSPROT;Acc: O15016] NP_597709.1 ENST00000331131 RAVER1 0.000164 1 5 [Source:RefSeq_peptide; Acc: NP_597709] ZFP36L1 ENST00000336440 Butyrateresponse factor 1 0.000169 1 5 (TIS11B protein) (EGF-response factor 1)(ERF-1). [Source: Uniprot/SWISSPROT; Acc: Q07352] U334_HUMANENST00000258258 Probable UPF0334 kinase-like 0.000183 1 7 protein.[Source: Uniprot/SWISSPROT; Acc: Q9BSD7] EHF ENST00000257831 etshomologous factor 0.000204 2 7 [Source: RefSeq_peptide; Acc: NP_036285]HMGB2 ENST00000296503 High mobility group protein 2 0.000243 1 6(HMG-2). [Source: Uniprot/SWISSPROT; Acc: P26583] CDKN2D ENST00000250245Cyclin-dependent kinase 4 0.000263 1 5 inhibitor D (p19-INK4d). [Source:Uniprot/SWISSPROT; Acc: P55273] CLDN5 ENST00000329916 Claudin-5(Transmembrane 0.000408 1 3 protein deleted in VCFS) (TMDVCF). [Source:Uniprot/SWISSPROT; Acc: O00501] EHHADH ENST00000231887 Peroxisomalbifunctional 0.000425 1 5 enzyme (PBE) (PBFE) [Includes: Enoyl-CoAhydratase (EC 4.2.1.17); 3,2-trans-enoyl-CoA isomerase (EC 5.3.3.8); 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35)]. [Source:Uniprot/SWISSPROT; Acc: Q08426] NP_115965.1 ENST00000253320lipopolysaccaride-specific 0.000453 2 1 response 5-like protein [Source:RefSeq_peptide; Acc: NP_115965] UPF3A ENST00000302685 Regulator ofnonsense 0.000455 1 4 transcripts 3A (Nonsense mRNA reducing factor 3A)(Up- frameshift suppressor 3 homolog A) (hUpf3). [Source:Uniprot/SWISSPROT; Acc: Q9H1J1] MSH4 ENST00000263187 MutS proteinhomolog 4. 0.000461 1 5 [Source: Uniprot/SWISSPROT; Acc: O15457]DPH5_HUMAN ENST00000251159 Probable diphthine synthase 0.000467 1 6 (EC2.1.1.98) (Diphthamide biosynthesis methyltransferase). [Source:Uniprot/SWISSPROT; Acc: Q9H2P9] TPK1 ENST00000360057 Thiaminpyrophosphokinase 1 0.000507 1 4 (EC 2.7.6.2) (Thiaminepyrophosphokinase 1) (hTPK1) (Placental protein 20) (PP20). [Source:Uniprot/SWISSPROT; Acc: Q9H3S4] NP_079217.1 ENST00000231526 hypotheticalprotein LOC321203 0.000514 1 4 [Source: RefSeq_peptide; Acc: NP_955832]hypothetical protein LOC321203 [Source: RefSeq_peptide; Acc: NP_955832]BY ORTHOLOGY TO: ENSDART00000046274 NP_065108.1 ENST00000179259PREDICTED: similar to 0.000517 2 3 chromosome 12 open reading frame 5[Source: RefSeq_peptide_predicted; Acc: XP_417232]PREDICTED: similar tochromosome 12 open reading frame 5 [Source: RefSeq_peptide_predicted;Acc: XP_417232] BY ORTHOLOGY TO: ENSGALT00000027940 NP_699185.1ENST00000296595 RIKEN cDNA 2810446P07 gene 0.00052 1 5 (2810446P07 Rik),mRNA [Source: RefSeq_dna; Acc: NM_175187] RIKEN cDNA 2810446P07 gene(2810446P07Rik), mRNA [Source: RefSeq_dna; Acc: NM_175187] BY ORTHOLOGYTO: ENSMUST00000057495 NP_068753.2 ENST00000336854 CG7053-PA 0.000525 14 [Source: RefSeq_peptide; Acc: NP_573326] CG7053-PA [Source:RefSeq_peptide; Acc: NP_573326] BY ORTHOLOGY TO: CG7053-RA CCT6AENST00000275603 T-complex protein 1, zeta 0.00053 1 4 subunit(TCP-1-zeta) (CCT-zeta) (CCT-zeta-1) (Tcp20) (HTR3) (Acute morphinedependence related protein 2). [Source: Uniprot/SWISSPROT; Acc: P40227]NP_057538.1 ENST00000267750 Hypothetical ORF. 0.000572 1 5 [Source:Saccharomyces Genome Database; Acc: S000003200]Hypothetical ORF.[Source: Saccharomyces Genome Database; Acc: S000003200] BY ORTHOLOGYTO: YGL231C NP_115678.1 ENST00000288607 PREDICTED: similar to RIKEN0.000591 1 5 cDNA 1810042K04 [Source: RefSeq_peptide_predicted; Acc:XP_213716]PREDICTED: similar to RIKEN cDNA 1810042K04 [Source:RefSeq_peptide_predicted; Acc: XP_213716] BY ORTHOLOGY TO:ENSRNOT00000001716 K0889_HUMAN ENST00000279034 PREDICTED: similar to0.000603 1 3 FLJ44670 protein [Source: RefSeq_peptide; Acc: XP_542984]PREDICTED: similar to FLJ44670 protein [Source: RefSeq_peptide; Acc:XP_542984] BY ORTHOLOGY TO: ENSCAFT00000013710 NP_937794.1ENST00000341723 expressed sequence AI987662 0.000616 1 5 [Source:MarkerSymbol; Acc: MGI: 2141520]expressed sequence AI987662 [Source:MarkerSymbol; Acc: MGI: 2141520] BY ORTHOLOGY TO: ENSMUST00000049985AEBP2 ENST00000266508 AE binding protein 2 0.00062 1 6 [Source:RefSeq_peptide; Acc: NP_694939] YWHAE ENST00000264335 14-3-3 proteinepsilon (14-3-3E). 0.000646 1 10 [Source: Uniprot/SWISSPROT; Acc:P62258] NP_659416.1 ENST00000281722 PREDICTED: similar to 0.000659 1 5hypothetical protein MGC27016 [Source: RefSeq_peptide_predicted; Acc:XP_227311]PREDICTED: similar to hypothetical protein MGC27016 [Source:RefSeq_peptide_predicted; Acc: XP_227311] BY ORTHOLOGY TO:ENSRNOT00000035846 NP_060818.3 ENST00000338099 PREDICTED: similar to0.000668 1 4 FLJ11171 protein [Source: RefSeq_peptide_predicted; Acc:XP_425111]PREDICTED: similar to FLJ11171 protein [Source:RefSeq_peptide_predicted; Acc: XP_425111] BY ORTHOLOGY TO:ENSGALT00000003716 PACE1_HUMAN ENST00000328079 Protein-associating withthe 0.000669 1 7 carboxyl-terminal domain of ezrin (Ezrin-bindingprotein PACE-1) (SCY1-like protein 3). [Source: Uniprot/SWISSPROT; Acc:Q8IZE3] HPN ENST00000337077 Serine protease hepsin (EC 0.000683 1 53.4.21.—) (Transmembrane protease, serine 1) [Contains: Serine proteasehepsin non- catalytic chain; Serine protease hepsin catalytic chain].[Source: Uniprot/SWISSPROT; Acc: P05981] CAP1 ENST00000340450 Adenylylcyclase-associated 0.00069 1 5 protein 1 (CAP 1). [Source:Uniprot/SWISSPROT; Acc: Q01518] MYO3A ENST00000265944 Myosin IIIA (EC2.7.1.37). 0.000701 1 4 [Source: Uniprot/SWISSPROT; Acc: Q8NEV4] DUSP8ENST00000331588 Dual specificity protein 0.000705 1 7 phosphatase 8 (EC3.1.3.48) (EC 3.1.3.16) (Dual specificity protein phosphatase hVH-5).[Source: Uniprot/SWISSPROT; Acc: Q13202] SPBC25 ENST00000282074 spindlepole body component 25 0.000709 2 3 [Source: RefSeq_peptide; Acc:NP_065726] NP_116218.1 ENST00000358906 PREDICTED: similar to 0.000712 15 hypothetical protein FLJ14721 [Source: RefSeq_peptide_predicted; Acc:XP_573412]PREDICTED: similar to hypothetical protein FLJ14721 [Source:RefSeq_peptide_predicted; Acc: XP_573412] BY ORTHOLOGY TO:ENSRNOT00000001588 NP_055764.2 ENST00000255016 RIKEN cDNA 2810403A07gene 0.000718 1 5 (2810403A07Rik), mRNA [Source: RefSeq_dna; Acc:NM_028814] RIKEN cDNA 2810403A07 gene (2810403A07Rik), mRNA [Source:RefSeq_dna; Acc: NM_028814] BY ORTHOLOGY TO: ENSMUST00000029696 ZFP64ENST00000216923 Zinc finger protein 64, isoforms 3 0.000745 1 4 and 4(Zinc finger protein 338). [Source: Uniprot/SWISSPROT; Acc: Q9NTW7]ZBTB37 ENST00000327714 Zinc finger and BTB domain 0.00079 1 4 containingprotein 37. [Source: Uniprot/SWISSPROT; Acc: Q5TC79] NP_004984.2ENST00000359819 ataxin 3 isoform 1 0.000796 2 6 [Source: RefSeq_peptide;Acc: NP_004984] SMS ENST00000362081 Spermine synthase (EC 0.000798 1 72.5.1.22) (Spermidine aminopropyltransferase) (SPMSY). [Source:Uniprot/SWISSPROT; Acc: P52788] MAML1 ENST00000292599 Mastermind-likeprotein 1 (Mam- 0.000799 1 3 1). [Source: Uniprot/SWISSPROT; Acc:Q92585] EDG3 ENST00000358157 Sphingosine 1-phosphate 0.000808 1 4receptor Edg-3 (S1P receptor Edg-3) (Endothelial differentationG-protein coupled receptor 3) (Sphingosine 1- phosphate receptor 3)(S1P3). [Source: Uniprot/SWISSPROT; Acc: Q99500] TESK2 ENST00000165317Dual specificity testis-specific 0.00081 1 5 protein kinase 2 (EC2.7.1.37) (EC 2.7.1.112) (Testicular protein kinase 2). [Source:Uniprot/SWISSPROT; Acc: Q96S53] BTBD2 ENST00000255608 BTB/POZ domaincontaining 0.000854 1 6 protein 2 [Source: Uniprot/SWISSPROT; Acc:Q9BX70] NP_076416.1 ENST00000285689 PREDICTED: similar to HCV 0.000865 14 NS3-transactivated protein 2 [Source: RefSeq_peptide; Acc: XP_531891]PREDICTED: similar to HCV NS3-transactivated protein 2 [Source:RefSeq_peptide; Acc: XP_531891] BY ORTHOLOGY TO: ENSCAFT00000000901Q5VWN5_HUMAN ENST00000263123 C10orf18 protein (Fragment). 0.00068 2 1[Source: Uniprot/SPTREMBL; Acc: Q6IPC8] DBT ENST00000260559 Lipoamideacyltransferase 0.000885 1 7 component of branched-chain alpha-keto aciddehydrogenase complex, mitochondrial precursor (EC 2.3.1.168)(Dihydrolipoyllysine-residue (2- methylpropanoyl)transferase) (E2)(Dihydrolipoamide branched chain transacylase) (BCKAD E2 [Source:Uniprot/SWISSPROT; Acc: P11162] EDAR ENST00000258443 Tumour necrosisfactor receptor 0.000886 1 4 superfamily member EDAR precursor(Anhidrotic ectodysplasin receptor 1) (Ectodysplasin-A receptor) (EDA-A1receptor) (Ectodermal dysplasia receptor) (Downless homolog). [Source:Uniprot/SWISSPROT; Acc: Q9UNE0] CTHRC1 ENST00000330295 Collagen triplehelix repeat- 0.000925 1 5 containing protein 1 precursor (NMTC1protein). [Source: Uniprot/SWISSPROT; Acc: Q96CG8] ZBT40_HUMANENST00000315432 Zinc finger and BTB domain 0.000952 1 5 containingprotein 40. [Source: Uniprot/SWISSPROT; Acc: Q9NUA8] NP_443089.2ENST00000361952 coiled-coil domain containing 16 0.000964 1 5 [Source:RefSeq_peptide; Acc: NP_443089] POU3F2 ENST00000328345 POU domain, class3, 0.000974 1 4 transcription factor 2 (Nervous- system specificoctamer-binding transcription factor N-Oct-3) (Brain-specifichomeobox/POU domain protein 2) (Brain-2) (Brn- 2 protein). [Source:Uniprot/SWISSPROT; Acc: P20265] PHF5A ENST00000216252 PHD finger-likedomain protein 0.000986 1 4 5A (Splicing factor 3B associated 14 kDaprotein) (SF3b14b). [Source: Uniprot/SWISSPROT; Acc: Q7RTV0] NP_115554.1ENST00000263997 solute carrier family 7, member 6 0.001062 1 4 oppositestrand [Source: RefSeq_peptide; Acc: NP_115554] SLC38A4 ENST00000266579solute carrier family 38, member 4 0.001065 1 4 [Source: RefSeq_peptide;Acc: NP_060488] MTM1 ENST00000306167 Myotubularin (EC 3.1.3.48).0.001094 1 4 [Source: Uniprot/SWISSPROT; Acc: Q13496] CDKN2BENST00000276925 Cyclin-dependent kinase 4 0.001131 2 4 inhibitor B(p14-INK4b) (p15- INK4b) (Multiple tumour suppressor 2) (MTS2). [Source:Uniprot/SWISSPROT; Acc: P42772] MIF ENST00000215754 Macrophage migrationinhibitory 0.00115 1 3 factor (MIF) (Phenylpyruvate tautomerase) (EC5.3.2.1) (Glycosylation-inhibiting factor) (GIF). [Source:Uniprot/SWISSPROT; Acc: P14174] ZCCHC8 ENST00000336229 Zinc finger CCHCdomain 0.001197 2 6 containing protein 8. [Source: Uniprot/SWISSPROT;Acc: Q6NZY4] ANKRD27 ENST00000306065 ankyrin repeat domain 27 (VPS90.001244 1 7 domain) [Source: RefSeq_peptide; Acc: NP_115515] OATENST00000224242 Ornithine aminotransferase, 0.001252 1 4 mitochondrialprecursor (EC 2.6.1.13) (Ornithine-oxo-acid aminotransferase) [Contains:Ornithine aminotransferase, hepatic form; Ornithine aminotransferase,renal form]. [Source: Uniprot/SWISSPROT; Acc: P04181] RNF13ENST00000344229 RING finger protein 13. 0.001254 2 7 [Source:Uniprot/SWISSPROT; Acc: O43567] TPIS_HUMAN ENST00000229270Triosephosphate isomerase (EC 0.001261 2 5 5.3.1.1) (TIM)(Triose-phosphate isomerase). [Source: Uniprot/SWISSPROT; Acc: P60174]CTNNB1 ENST00000349496 Beta-catenin. 0.001303 1 10 [Source:Uniprot/SWISSPROT; Acc: P35222] CX041_HUMAN ENST00000161782 ProteinCXorf41 (Sarcoma 0.001385 1 4 antigen NY-SAR-97). [Source:Uniprot/SWISSPROT; Acc: Q9NQM4] BUB1B ENST00000287598 Mitotic checkpoint0.001399 2 5 serine/threonine-protein kinase BUB1 beta (EC 2.7.1.37)(hBUBR1) (MAD3/BUB1-related protein kinase) (Mitotic checkpoint kinaseMAD3L) (SSK1). [Source: Uniprot/SWISSPROT; Acc: O60566] OSR1ENST00000272223 odd-skipped related 1 0.001413 1 6 [Source:RefSeq_peptide; Acc: NP_660303] GPR88 ENST00000318647 Probable G-proteincoupled 0.001437 1 4 receptor 88 (Striatum-specific G- protein coupledreceptor). [Source: Uniprot/SWISSPROT; Acc: Q9GZN0] CCNB3ENST00000276014 G2/mitotic-specific cyclin B3. 0.001489 1 5 [Source:Uniprot/SWISSPROT; Acc: Q8WWL7] PYGM ENST00000164139 Glycogenphosphorylase, 0.001511 1 5 muscle form (EC 2.4.1.1) (Myophosphorylase).[Source: Uniprot/SWISSPROT; Acc: P11217] BNIP2 ENST00000267859BCL2/adenovirus E18 19-kDa 0.001591 1 6 protein-interacting protein 2.[Source: Uniprot/SWISSPROT; Acc: Q12982] YAP1 ENST00000282441 65 kDaYes-associated protein 0.001635 1 5 (YAP65). [Source: Uniprot/SWISSPROT;Acc: P46937] NP_079001.2 ENST00000278520 Adult male tongue cDNA, RIKEN0.001669 1 4 full-length enriched library, clone: 2310043N13 product:weakly similar to HT025 (9 days embryo whole body cDNA, RIKENfull-length enriched library, clone: D030052K04 product: weakly similarto HT025). [Source: Uniprot/SPTREMBL; Acc: Q9D6Z7]Adult male tonguecDNA, RIKEN full-length enriched library, clone: 2310043N13 product:weakly similar to HT025 (9 days embryo whole body cDNA, RIKENfull-length enriched library, clone: D030052K04 product: weakly similarto HT025). [Source: Uniprot/SPTREMBL; Acc: Q9D6Z7] BY ORTHOLOGY TO:ENSMUST00000067578 NP_065988.1 ENST00000267430 Fanconi anemia, 0.0016922 2 complementation group M [Source: RefSeq_peptide; Acc: NP_065988]MIDN ENST00000300952 midnolin 0.001695 1 4 [Source: RefSeq_peptide; Acc:NP_796375] NP_963840.2 ENST00000287899 similar to NADH-cytochrome b50.001761 1 5 reductase [Source: RefSeq_peptide; Acc: XP_396073] similarto NADH- cytochrome b5 reductase [Source: RefSeq_peptide; Acc:XP_396073] BY ORTHOLOGY TO: ENSAPMT00000013057 NXPH1 ENST00000265579Neurexophilin-1 precursor. 0.001878 2 6 [Source: Uniprot/SWISSPROT; Acc:P58417] ZIC2 ENST00000245295 Zinc finger protein ZIC 2 (Zinc 0.001906 16 finger protein of the cerebellum 2). [Source: Uniprot/SWISSPROT; Acc:O95409] ERMAP ENST00000328249 erythroblast membrane- 0.001966 2 1associated protein [Source: RefSeq_peptide; Acc: NP_001017922] IRX4ENST00000231357 Iroquois-class homeodomain 0.002018 1 3 protein IRX-4(Iroquois homeobox protein 4) (Homeodomain protein IRXA3). [Source:Uniprot/SWISSPROT; Acc: P78413] PNOC ENST00000301908 Nociceptinprecursor [Contains: 0.002057 1 4 Neuropeptide 1; Nociceptin (OrphaninFQ) (PPNOC); Neuropeptide 2]. [Source: Uniprot/SWISSPROT; Acc: Q13519]ZNF583 ENST00000291598 zinc finger protein 583 0.00212 1 4 [Source:RefSeq_peptide; Acc: NP_689691] CCND1 ENST00000227507 G1/S-specificcyclin D1 (PRAD1 0.002183 1 4 oncogene) (BCL-1 oncogene). [Source:Uniprot/SWISSPROT; Acc: P24385] NP_116316.1 ENST00000254742 RIKEN cDNA2810021O14 gene 0.002194 2 5 (2810021O14Rik), mRNA [Source: RefSeq_dna;Acc: NM_025480] RIKEN cDNA 2810021O14 gene (2810021O14Rik), mRNA[Source: RefSeq_dna; Acc: NM_025480] BY ORTHOLOGY TO: ENSMUST00000087511RNF125 ENST00000217740 RING finger protein 125 (EC 0.002203 1 4 6.3.2.—)(T-cell RING activation protein 1) (TRAC-1). [Source: Uniprot/SWISSPROT;Acc: Q96EQ8] TRPC6 ENST00000344327 Short transient receptor potential0.002295 1 4 channel 6 (TrpC6). [Source: Uniprot/SWISSPROT; Acc: Q9Y210]NPEPPS ENST00000322157 Puromycin-sensitive 0.002348 1 8 aminopeptidase(EC 3.4.11.—) (PSA). [Source: Uniprot/SWISSPROT; Acc: P55786]Q9ULK9_HUMAN ENST00000264229 PREDICTED: similar to 0.002364 1 4Nucleolar phosphoprotein p130 (Nucleolar 130 kDa protein) (140 kDanucleolar phosphoprotein) (Nopp140) (Nucleolar and coiled-bodyphosphoprotein 1) [Source: RefSeq_peptide_predicted; Acc:XP_214022]PREDICTED: similar to Nucleolar phosphoprotein p130 (Nucleolar130 kDa protein) (140 kDa nucleolar phosphoprotein) (Nopp140) (Nucleolarand coiled-body phosphoprotein 1) [Source: RefSeq_peptide_predicted;Acc: XP_214022] BY ORTHOLOGY TO: ENSRNOT00000002908 GFRA2ENST00000306793 GDNF family receptor alpha 2 0.002495 1 6 precursor(GFR-alpha 2) (Neurturin receptor alpha) (NTNR-alpha) (NRTNR-alpha)(TGF-beta-related neurotrophic factor receptor 2) (GDNF receptor beta)(GDNFR-beta) (RET ligand 2). [Source: Uniprot/SWISSPROT; Acc: O00451]MTHFD2 ENST00000264090 Bifunctional 0.002509 1 5methylenetetrahydrofolate dehydrogenase/cyclohydrolase, mitochondrialprecursor [Includes: NAD-dependent methylenetetrahydrofolatedehydrogenase (EC 1.5.1.15); Methenyltetrahydrofolate cyclohydrolase (EC3.5.4.9)]. [Source: Uniprot/SWISSPROT; Acc: P13995] TEAD2ENST00000311227 Transcriptional enhancer factor 0.002529 1 3 TEF-4 (TEAdomain family member 2) (TEAD-2). [Source: Uniprot/SWISSPROT; Acc:Q15562] YT521_HUMAN ENST00000355665 Putative splicing factor YT521.0.002572 2 1 [Source: Uniprot/SWISSPROT; Acc: Q96MU7] TBX1ENST00000332710 T-box transcription factor TBX1 0.002675 1 6 (T-boxprotein 1) (Testis-specific T-box protein). [Source: Uniprot/SWISSPROT;Acc: O43435] Q86X61_HUMAN ENST00000334227 OVOS2 protein. 0.00268 2 2[Source: Uniprot/SPTREMBL; Acc: Q86X61] EVL ENST00000355449Ena/vasodilator stimulated 0.00269 2 3 phosphoprotein-like protein(Ena/VASP-like protein). [Source: Uniprot/SWISSPROT; Acc: Q9UI08] EWSR1ENST00000332050 RNA-binding protein EWS (EWS 0.002703 2 1 oncogene)(Ewing sarcoma breakpoint region 1 protein). [Source: Uniprot/SWISSPROT;Acc: Q01844] MMRN2 ENST00000310615 Multimerin 2 precursor (EMILIN-0.002759 1 4 3) (Elastin microfibril interface located protein 3)(Elastin microfibril interfacer 3) (EndoGlyx-1 p125/p140 subunit).[Source: Uniprot/SWISSPROT; Acc: Q9H8L6] FNTA ENST00000302279 Protein0.002813 1 6 farnesyltransferase/geranylgeranyltransferase type I alphasubunit (EC 2.5.1.58) (EC 2.5.1.59) (CAAX farnesyltransferase alphasubunit) (Ras proteins prenyltransferase alpha) (FTase- alpha) (Type Iprotein geranyl- geranyltransferase alpha subunit) [Source:Uniprot/SWISSPROT; Acc: P49354] OMD ENST00000247535 Osteomodulinprecursor 0.002819 1 3 (Osteoadherin) (OSAD) (Keratan sulfateproteoglycan osteomodulin) (KSPG osteomodulin). [Source:Uniprot/SWISSPROT; Acc: Q99983] ZNF654 ENST00000309495 zinc fingerprotein 654 0.002862 2 3 [Source: RefSeq_peptide; Acc: NP_060763] ING3ENST00000315883 Inhibitor of growth protein 3 0.003065 2 1 (p47ING3protein). [Source: Uniprot/SWISSPROT; Acc: Q9NXR8] Q9UHS9_HUMANENST00000357012 NOT ANNOTATED 0.003127 2 2 DRG2 ENST00000225729Developmentally regulated GTP- 0.003145 1 4 binding protein 2 (DRG 2).[Source: Uniprot/SWISSPROT; Acc: P55039] NP_116203.1 ENST00000257575Putative protein, with at least 4 0.003156 1 4 transmembrane domains, ofbilaterial origin (38.9 kD) (XB497) [Source:; Acc: Cel.9253]Putativeprotein, with at least 4 transmembrane domains, of bilaterial origin(38.9 kD) (XB497) [Source:; Acc: Cel.9253] BY ORTHOLOGY TO: ZC13.1bNP_849149.2 ENST00000324698 RIKEN cDNA 4932408B21 gene 0.003163 1 4(4932408B21Rik), mRNA [Source: RefSeq_dna; Acc: NM_172535] RIKEN cDNA4932408B21 gene (4932408B21Rik), mRNA [Source: RefSeq_dna; Acc:NM_172535] BY ORTHOLOGY TO: ENSMUST00000052277 GPR85 ENST00000297146Probable G-protein coupled 0.003165 1 6 receptor 85 (Super conservedreceptor expressed in brain 2). [Source: Uniprot/SWISSPROT; Acc: P60893]ENST00000266432 NOT ANNOTATED 0.003275 2 1 SDFR1 ENST00000345330 stromalcell derived factor 0.003289 1 9 receptor 1 isoform a [Source:RefSeq_peptide; Acc: NP_059429] ARMC4 ENST00000239715 armadillo repeatcontaining 4 0.003311 2 1 [Source: RefSeq_peptide; Acc: NP_060546] ATF2ENST00000295499 Cyclic-AMP-dependent 0.003334 2 1 transcription factorATF-2 (Activating transcription factor 2) (cAMP response element bindingprotein CRE-BP1) (HB16). [Source: Uniprot/SWISSPROT; Acc: P15336]Q9P0E8_HUMAN ENST00000330598 NOT ANNOTATED 0.00338 2 1 PANX1ENST00000227638 Pannexin-1. 0.003393 2 5 [Source: Uniprot/SWISSPROT;Acc: Q96RD7] XP_068632.2 ENST00000274299 PREDICTED: hypothetical0.003394 1 3 protein XP_068632 [Source: RefSeq_peptide_predicted; Acc:XP_068632] GALK2 ENST00000327171 N-acetylgalactosamine kinase 0.003406 25 (EC 2.7.1.—) (GalNAc kinase) (Galactokinase 2). [Source:Uniprot/SWISSPROT; Acc: Q01415] NP_071903.2 ENST00000347571 limb region1 protein 0.003545 2 1 [Source: RefSeq_peptide; Acc: NP_071903]NP_061961.2 ENST00000221265 Paf1, RNA polymerase II 0.003601 1 4associated factor, homolog [Source: RefSeq_peptide; Acc: NP_061961]EVG1_HUMAN ENST00000249079 Hypothetical UPF0193 protein 0.003609 2 5EVG1 homolog. [Source: Uniprot/SWISSPROT; Acc: Q9VSS7]HypotheticalUPF0193 protein EVG1 homolog. [Source: Uniprot/SWISSPROT; Acc: Q9VSS7]BY ORTHOLOGY TO: CG5280-RA GMPR ENST00000259727 GMP reductase 1 (EC1.7.1.7) 0.003624 1 4 (Guanosine 5′-monophosphate oxidoreductase 1)(Guanosine monophosphate reductase 1). [Source: Uniprot/SWISSPROT; Acc:P36959] CBR1 ENST00000290349 Carbonyl reductase [NADPH] 1 0.003639 1 3(EC 1.1.1.184) (NADPH- dependent carbonyl reductase 1)(Prostaglandin-E(2) 9- reductase) (EC 1.1.1.189) (Prostaglandin9-ketoreductase) (15-hydroxyprostaglandin dehydrogenase [NADP+]) (EC1.1.1.197). [Source: Uniprot/SWISSPROT; Acc: P16152] F10C1_HUMANENST00000277884 Protein FRA10AC1. 0.003645 2 3 [Source:Uniprot/SWISSPROT; Acc: Q70Z53] FAM13C1 ENST00000277705 Protein FAM13C1.0.003648 1 4 [Source: Uniprot/SWISSPROT; Acc: Q8NE31] EPB41L5ENST00000331393 Band 4.1-like protein 5. 0.00369 2 1 [Source:Uniprot/SWISSPROT; Acc: Q9HCM4] IL5 ENST00000231454 Interleukin-5precursor (IL-5) (T- 0.003712 1 4 cell replacing factor) (TRF)(Eosinophil differentiation factor) (B cell differentiation factor I).[Source: Uniprot/SWISSPROT; Acc: P05113] NP_115676.1 ENST00000312777trichoplein 0.003739 1 4 [Source: RefSeq_peptide; Acc: NP_115676]NP_660308.1 ENST00000296824 RIKEN cDNA 0610011N22 gene 0.003745 1 3(0610011N22Rik), mRNA [Source: RefSeq_dna; Acc: NM_024201] RIKEN cDNA0610011N22 gene (0610011N22Rik), mRNA [Source: RefSeq_dna; Acc:NM_024201] BY ORTHOLOGY TO: ENSMUST00000022063 TAS2R14 ENST00000240689Taste receptor type 2 member 0.003749 2 2 14 (T2R14) (Taste receptorfamily B member 1) (TRB1). [Source: Uniprot/SWISSPROT; Acc: Q9NYV8]SFRS10 ENST00000342294 Arginine/serine-rich splicing 0.003752 2 1 factor10 (Transformer-2-beta) (HTRA2-beta) (Transformer 2 protein homolog).[Source: Uniprot/SWISSPROT; Acc: P62995] AKT1 ENST00000310523 RAC-alphaserine/threonine- 0.003758 1 4 protein kinase (EC 2.7.1.37)(RAC-PK-alpha) (Protein kinase B) (PKB) (C-AKT). [Source:Uniprot/SWISSPROT; Acc: P31749] WTAP ENST00000337387 Wilms' tumour1-associating 0.003802 2 1 protein (WT1-associated protein) (Putativepre-mRNA splicing regulator female-lethal(2D) homolog). [Source:Uniprot/SWISSPROT; Acc: Q15007] HTATIP2 ENST00000338653 HIV-1 Tatinteractive protein 2, 0.003824 1 6 30 kDa [Source: RefSeq_peptide; Acc:NP_006401] ENST00000313360 NOT ANNOTATED 0.003887 2 2 KLF6ENST00000173785 Core promoter element-binding 0.00389 1 5 protein(Kruppel-like factor 6) (B- cell derived protein 1) (Proto- oncogeneBCD1) (Transcription factor Zf9) (GC-rich sites binding factor GBF).[Source: Uniprot/SWISSPROT; Acc: Q99612] Q5VWN5_HUMAN ENST00000328090C10orf18 protein (Fragment). 0.003926 2 2 [Source: Uniprot/SPTREMBL;Acc: Q6IPC8] CERK ENST00000216264 Ceramide kinase (EC 2.7.1.138) 0.003931 4 (Acylsphingosine kinase) (hCERK) (Lipid kinase 4) (LK4). [Source:Uniprot/SWISSPROT; Acc: Q8TCT0] HSPA5 ENST00000265959 78 kDaglucose-regulated 0.003975 1 5 protein precursor (GRP 78)(Immunoglobulin heavy chain binding protein) (BiP) (Endoplasmicreticulum lumenal Ca(2+) binding protein grp78). [Source:Uniprot/SWISSPROT; Acc: P11021] KLHL1 ENST00000279889 Kelch-likeprotein 1. 0.004008 2 5 [Source: Uniprot/SWISSPROT; Acc: Q9NR64] RPSAENST00000301821 40S ribosomal protein SA (p40) 0.004055 1 3 (34/67 kDalaminin receptor) (Colon carcinoma laminin- binding protein) (NEM/1CHD4)(Multidrug resistance- associated protein MGr1-Ag). [Source:Uniprot/SWISSPROT; Acc: P08865] TBC1D1 ENST00000261439 TBC1 domainfamily member 1. 0.004086 2 5 [Source: Uniprot/SWISSPROT; Acc: Q86TI0]GPR109A ENST00000328880 Probable G-protein coupled 0.00416 1 3 receptor109B (G-protein coupled receptor HM74). [Source: Uniprot/SWISSPROT; Acc:P49019] ATN1 ENST00000356654 Atrophin-1 (Dentatorubral- 0.004207 1 4pallidoluysian atrophy protein). [Source: Uniprot/SWISSPROT; Acc:P54259] O95036_HUMAN ENST00000304230 NOT ANNOTATED 0.004216 2 1 SLC39A8ENST00000265515 solute carrier family 39 (zinc 0.00427 1 5 transporter),member 8 [Source: RefSeq_peptide; Acc: NP_071437] NP_054890.1ENST00000238892 postsynaptic protein CRIPT 0.004295 2 4 [Source:RefSeq_peptide; Acc: NP_054890] CDKN2C ENST00000262662 Cyclin-dependentkinase 6 0.004302 1 3 inhibitor (p18-INK6) (Cyclin- dependent kinase 4inhibitor C) (p18-INK4c). [Source: Uniprot/SWISSPROT; Acc: P42773] NUPL1ENST00000306113 Nucleoporin p58/p45 0.004345 1 5 (Nucleoporin-likeprotein 1). [Source: Uniprot/SWISSPROT; Acc: Q9BVL2] CJ082_HUMANENST00000356850 NOT ANNOTATED 0.004356 2 1 ELK1 ENST00000247161 ETSdomain protein Elk-1. 0.004382 1 4 [Source: Uniprot/SWISSPROT; Acc:P19419] CENTD3 ENST00000239440 Centaurin-delta 3 (Cnt-d3) (Arf- 0.0043841 6 GAP, Rho-GAP, ankyrin repeat and pleckstrin homologydomains-containing protein 3). [Source: Uniprot/SWISSPROT; Acc: Q8WWN8]NP_005109.2 ENST00000313163 tumour necrosis factor (ligand) 0.00447 1 4superfamily, member 15 [Source: RefSeq_peptide; Acc: NP_005109]Q5VY36_HUMAN ENST00000258651 OTTHUMP00000018470. 0.004492 2 2 [Source:Uniprot/SPTREMBL; Acc: Q5VY36] CDH8 ENST00000299345 Cadherin-8precursor. 0.004507 1 6 [Source: Uniprot/SWISSPROT; Acc: P55286] CD99L2ENST00000320893 CD99 antigen-like 2 isoform E3′- 0.004543 2 1 E4′-E3-E4[Source: RefSeq_peptide; Acc: NP_113650] PMP22 ENST00000312280Peripheral myelin protein 22 0.004582 1 9 (PMP-22). [Source:Uniprot/SWISSPROT; Acc: Q01453] KIAA1958 ENST00000356558 PREDICTED:similar to RIKEN 0.004632 1 7 cDNA E130308A19 [Source: RefSeq_peptide;Acc: XP_538791] PREDICTED: similar to RIKEN cDNA E130308A19 [Source:RefSeq_peptide; Acc: XP_538791] BY ORTHOLOGY TO: ENSCAFT00000004894LECT1 ENST00000258609 Chondromodulin-I precursor 0.004669 1 7 (ChM-I)(Leukocyte cell-derived chemotaxin 1) [Contains: Chondrosurfactantprotein (CH- SP)]. [Source: Uniprot/SWISSPROT; Acc: O75829]NP_001003684.1 ENST00000332801 ubiquinol-cytochrome c 0.004744 1 2reductase complex 7.2 kDa protein isoform b [Source: RefSeq_peptide;Acc: NP_001003684] PDXK ENST00000291565 Pyridoxal kinase (EC 2.7.1.35)0.004927 1 3 (Pyridoxine kinase). [Source: Uniprot/SWISSPROT; Acc:O00764] PDC ENST00000271587 Phosducin (PHD) (33 kDa 0.004942 1 5phototransducing protein) (MEKA protein). [Source: Uniprot/SWISSPROT;Acc: P20941] XPO1 ENST00000195419 Exportin-1 (Chromosome region 0.0049471 7 maintenance 1 protein homolog). [Source: Uniprot/SWISSPROT; Acc:O14980] FZD5 ENST00000295417 Frizzled 5 precursor (Frizzled-5) 0.0049851 5 (Fz-5) (hFz5) (FzE5). [Source: Uniprot/SWISSPROT; Acc: Q13467]Q8WV45_HUMAN ENST00000307544 Novel protein. 0.005014 1 1 [Source:Uniprot/SPTREMBL; Acc: Q5T5P3] NUP37 ENST00000251074 Nucleoporin Nup37(p37). 0.005016 1 3 [Source: Uniprot/SWISSPROT; Acc: Q8NFH4] TAF11ENST00000334316 Transcription initiation factor 0.00503 1 4 TFIIDsubunit 11 (Transcription initiation factor TFIID 28 kDa subunit)(TAF(II)28) (TAFII-28) (TAFII28) (TFIID subunit p30- beta). [Source:Uniprot/SWISSPROT; Acc: Q15544] APBA3 ENST00000316757 Amyloid beta A4precursor 0.005054 1 5 protein-binding family A member 3(Neuron-specific X11L2 protein) (Neuronal Munc18-1- interacting protein3) (Mint-3) (Adapter protein X11gamma). [Source: Uniprot/SWISSPROT; Acc:O96018] NP_689672.2 ENST00000315997 PREDICTED: similar to 0.00519 1 5MGC45438 protein [Source: RefSeq_peptide_predicted; Acc:XP_155973]PREDICTED: similar to MGC45438 protein [Source:RefSeq_peptide_predicted; Acc: XP_155973] BY ORTHOLOGY TO:ENSMUST00000050160 NP_065170.1 ENST00000215906 PREDICTED: similar to0.0052 1 4 aspartyl(asparaginyl)beta- hydroxylase; HAAH [Source:RefSeq_peptide_predicted; Acc: XP_424691]PREDICTED: similar toaspartyl(asparaginyl)beta- hydroxylase; HAAH [Source:RefSeq_peptide_predicted; Acc: XP_424691] BY ORTHOLOGY TO:ENSGALT00000004159 Q96LP0_HUMAN ENST00000299326 NOT ANNOTATED 0.005387 12 Q9P2J0_HUMAN ENST00000258005 NHS-like 1 0.005482 1 4 [Source:MarkerSymbol; Acc: MGI: 106390]NHS-like 1 [Source: MarkerSymbol; Acc:MGI: 106390] BY ORTHOLOGY TO: ENSMUST00000037341 CLP24_HUMANENST00000253934 Claudin-like protein 24. 0.005535 1 4 [Source:Uniprot/SWISSPROT; Acc: Q9BSN7] SBF2 ENST00000256190 SET binding factor2 0.005559 1 2 [Source: RefSeq_peptide; Acc: NP_112224] BRP44LENST00000341756 Brain protein 44-like protein. 0.005595 1 6 [Source:Uniprot/SWISSPROT; Acc: Q9Y5U8] EEF1E1 ENST00000264871 Eukaryotictranslation elongation 0.005618 1 5 factor 1 epsilon-1 (Multisynthetasecomplex auxiliary component p18) (Elongation factor p18). [Source:Uniprot/SWISSPROT; Acc: O43324] NP_036404.1 ENST00000261897 Huntingtininteracting protein C 0.005628 1 3 isoform 2 [Source: RefSeq_peptide;Acc: NP_036404] KLRC1 ENST00000347831 NKG2-A/NKG2-B type II integral0.005685 1 1 membrane protein (NKG2-A/B activating NK receptor) (NK cellreceptor A). [Source: Uniprot/SWISSPROT; Acc: P26715] NP_694946.1ENST00000331203 PREDICTED: similar to 0.005686 1 5 hypothetical proteinFLJ37440 [Source: RefSeq_peptide_predicted; Acc: XP_419304]PREDICTED:similar to hypothetical protein FLJ37440 [Source:RefSeq_peptide_predicted; Acc: XP_419304] BY ORTHOLOGY TO:ENSGALT00000013441 KLRC1 ENST00000359151 NKG2-A/NKG2-B type II integral0.005697 1 2 membrane protein (NKG2-A/B activating NK receptor) (NK cellreceptor A). [Source: Uniprot/SWISSPROT; Acc: P26715] NP_997343.1ENST00000340819 NOT ANNOTATED 0.005703 1 2 SUPT16H ENST00000216297chromatin-specific transcription 0.005743 1 6 elongation factor largesubunit [Source: RefSeq_peptide; Acc: NP_009123] MUC16 ENST00000331986Ovarian cancer related tumour 0.005825 1 2 marker CA125. [Source:Uniprot/SPTREMBL; Acc: Q8WXI7] NP_689988.1 ENST00000307588 KM-HN-1protein 0.005895 1 3 [Source: RefSeq_peptide; Acc: NP_689988]TRA2A_HUMAN ENST00000297071 Transformer-2 protein homolog 0.005906 1 6(TRA-2 alpha). [Source: Uniprot/SWISSPROT; Acc: Q13595] NP_077001.1ENST00000319285 RIKEN cDNA 2410015N17 gene 0.005907 1 3 (2410015N17Rik),mRNA [Source: RefSeq_dna; Acc: NM_023203] RIKEN cDNA 2410015N17 gene(2410015N17Rik), mRNA [Source: RefSeq_dna; Acc: NM_023203] BY ORTHOLOGYTO: ENSMUST00000035276 SFRS11 ENST00000235399 Splicing factorarginine/serine- 0.005937 1 7 rich 11 (Arginine-rich 54 kDa nuclearprotein) (p54). [Source: Uniprot/SWISSPROT; Acc: Q05519] CALN1ENST00000329008 Calneuron-1 (Calcium-binding 0.00597 1 7 protein CaBP8).[Source: Uniprot/SWISSPROT; Acc: Q9BXU9] NP_078959.2 ENST00000306049RIKEN cDNA 1110002N22 gene 0.005972 1 3 (1110002N22Rik), mRNA [Source:RefSeq_dna; Acc: NM_183275] RIKEN cDNA 1110002N22 gene (1110002N22Rik),mRNA [Source: RefSeq_dna; Acc: NM_183275] BY ORTHOLOGY TO:ENSMUST00000050207 ATP6V1G1 ENST00000259415 Vacuolar ATP synthasesubunit 0.005978 2 4 G 1 (EC 3.6.3.14) (V-ATPase G subunit 1) (Vacuolarproton pump G subunit 1) (V-ATPase 13 kDa subunit 1) (Vacuolar ATPsynthase subunit M16). [Source: Uniprot/SWISSPROT; Acc: O75348] DRP2ENST00000263029 Dystrophin-related protein 2. 0.005999 1 3 [Source:Uniprot/SWISSPROT; Acc: Q13474]

Example 6

Cluster Analysis of miRNA Expression in Mouse Lung Specimens

Total RNA from mouse normal and tumor tissues was prepared using theTrizol reagent (Invitrogen, CA). The microarray has LNA-modifiedoligonucleotide probes to all annotated miRNAs from the mouse (313miRNAs) and humans (238 miRNAs) in the miRBase MicroRNA database Release7.1. These miRNAs are displayed in quadruplicate independent positionson each array. The arrays also contain appropriate positive and negativecontrols, as purchased from Exigon A/S (Copenhagen, Denmark). Each arraywas independently hybridized two or more times to assure results arereplicated. Interpretable findings require concordant findings of fourpresentations of individual miRNAs. Labeled RNA was hybridized overnightat 65° C. in a hybridization mixture containing 4×SSC, 0.1% SDS, 1 μg/μlHerring Sperm DNA and 38% formamide. Hybridized slides were washed threetimes in 2×SSC, 0.25% SDS at 65° C., followed by three times in0.08×SSC, and finally three times in 0.4×SSC at room temperature. Themicroarrays were scanned with the ArrayWorx scanner (Applied Precision,USA) and manufacturer's procedures. Scanned images were imported intoTIGR Spot Finder version 3.1 (50) for the extraction of mean spotintensities and median local background intensities excluding spots withintensities below median local background and +4× standard deviations.Background-correlated intensities were normalized using variancestabilizing normalization package version 1.8.0 (51) for R (The RProject for Statistical Computing). Intensities of replicate spots areaveraged using Microsoft Excel. Probes displaying a coefficient ofvariance >100% are excluded from further analyses. Results are displayedin FIG. 5.

Example 7 Semi-Quantitative RT-PCR Assays

Total RNA was isolated from desired human and mouse lung tissues asabove. Individual miRNA species were detected using the mirVana™ qRT-PCRmiRNA detection procedure (Ambion, Tx). This procedure allowsnonisotopic, sensitive, and rapid quantitation of mature microRNA(miRNA) expression levels. 25 ng of total RNA was reverse transcribedusing a miR-34c specific primer including a minus reverse transcriptasecontrol. PCR was then carried out according to the manufacturersinstructions. The resulting approximately 90 bp PCR products wereresolved on a 3% agarose gel and stained with GelStar nucleic acid gelstain (Cambrex, Me.). Results are displayed in FIG. 6.

Example 8

Over-Expression of miRNAs in Murine Transgenic Lung Cancer

Comprehensive locked nucleic acid (LNA) microarray analyses wereconducted to profile miRNA expression independently in malignant andnormal lung tissues isolated from human surfactant protein C(SP-C)driven wild-type and proteasome-degradation resistant cyclin Etransgenic lines (Ma et al). Degradation-resistant cyclin E lines hadincreased neoplastic lesions as compared to wild-type cyclin E lineseven when cyclin E expression levels were comparable (Ma at al).Adenocarcinomas and adjacent normal lung tissues from these transgeniclines and normal lung tissues from age and sex matched non-transgenic(Tg−) FVB mice were each examined in these miRNA microarrays containing315 murine miRNAs. FIG. 8A displays statistically significantlyexpression changes of the most to least over-expressed miRNAs. FIG. 8Bpresents the fold changes of these miRNAs relative to Tg− normal lungtissues.

These lung cancers had 114 to 4 fold higher expression levels of thehighlighted miRNAs as compared to Tg− normal lung tissues. Three miRNAswith highest expression levels (>10 fold) in these lung cancers were:miR-136, miR-376a and miR-31, as shown in FIG. 8B. All three miRNAs werepreviously unrecognized as highlighted miRNAs in lung cancer.

Transgenic Lung Tissues

Murine cyclin E transgenic lines that exhibit pre-malignant andmalignant (adenocarcinoma) lung lesions were previously described (Ma etal). Those studied here included human SP-C-driven wild-type cyclin Etransgenic (line 2) and proteasome degradation-resistant (line 4) lines(18). Adenocarcinomas and adjacent histopathologically normal lungtissues were individually harvested from age and sex matched mice andimmediately placed in RNAlater (Ambion, Austin, Tex.). Total RNA wasisolated using established techniques for miRNA expression arrays.Formalin-fixed and paraffin-embedded transgenic lung tissues wereharvested and used for ISH assays.

RNA Isolation and miRNA Arrays

The RNA isolation and miRNA array procedures were described previously(Lui at al). In brief, total RNA was isolated from cell lines and lungtissues with TRIzol reagent (Invitrogen, Carlsbad, Calif.) and was 3′end labeled using T4 RNA ligase to couple Cy3-labeled RNA linkers.Labeled RNA was hybridized to LNA microarrays overnight at 65° C. in ahybridization mixture containing 4× sodium chloride citrate (SSC)(1×SSC: 150 mM sodium chloride and 15 mM sodium citrate), 0.1% SDS, 1μg/μl herring sperm DNA and 38% formamide. Slides were washed threetimes in 2×SSC, 0.025% SDS at 65° C., three times in 0.8×SSC and threetimes in 0.4×SSC at room temperature. Each RNA sample was independentlyhybridized twice. There were four probe sets used for each miRNA. Onlyconcordant hybridization results were scored. Microarrays were scannedusing an ArrayWorx scanner (Applied Precision, Issaquah, Wash.). Imageswere analyzed using GridGrinder (http COLON SLASH SLASH gridgrinder DOTsourceforge DOT net/) and background-subtracted spot intensities werenormalized using variance stabilization normalization (Sempere et al).The miRNAs selected for in-depth study showed statistically significantexpression differences in both murine and human normal versus malignantlung tissues.

Validation Using ISH Assays

To independently validate and to determine spatial distribution patternsof specific miRNAs, ISH assays were used. As shown in FIG. 9, malignant(adenocarcinoma) and adjacent normal lung tissues were studied intransgenic cyclin E mice. Histopathologic analyses of lung tissues wereperformed to identify normal and malignant lung tissues. Expressionprofiles of miR-31 (the functionally highlighted miRNA), miR-21 andcontrol 18S rRNA were each analyzed by ISH in malignant and normal lungtissues from transgenic mice. As displayed, miR-31 exhibited reducedminimal expression in normal lung tissue, but high miR-31 expression wasdetected within malignant lung tissue. Concordant results were observedin human paired normal-malignant lung tissues, as in FIG. 9. Low levelsof miR-31 expression whereas found in normal human lung tissue, but ahigh expression level was present in malignant lung tissue. Asanticipated from prior work miR-21 was also detected in FIG. 9 at higherexpression levels in malignant than in normal human lung tissue. The 18SrRNA signal was ubiquitously expressed in both normal and malignant lungtissues, confirming integrity of RNA used in these tissue analyses.Together, these results confirm expression patterns of specific miRNAsin malignant versus normal lung tissues.

In Situ Hybridization Assays

ISH assays were performed according to standard procedures as describedin Example 8B) above. In brief, slides were prehybridized inhybridization solution (50% formamide, 5% SSC, 500 μg/mL yeast tRNA, and1% Denhardt's solution) at 50° C. for 30 min. Ten pmol of the desiredFITC-labeled, LNA-modified DNA probes (Integrated DNA Technologies,Coralville, Iowa) complementary to specific miRNAs and/or biotinylatedunmodified DNA probes against 18S rRNA were added and hybridized for 2hours at a temperature 20° C. to 25° C. below the calculated meltingtemperature of the LNA probe. After stringent washes, a tyramide signalamplification (TSA) reaction was carried out using the GenPointFluorescein kit (DakoCytomation, Glostrup, Denmark) and themanufacturer's recommended procedures and with the substitution ofstreptavidin/HRP (Invitrogen, Carlsbad, Calif.) for detection ofbiotinylated probes. A standard immunohistochemistry protocol was usedto detect cytokeratin 19 (CK19 1:200 dilution; Biogenex, San Ramon,Calif.) and revealed by TSA with secondary anti-mouse/HRP (1:500dilution; Biorad, Hercules, Calif.). Slides were mounted with ProlongGold solution (Invitrogen).

Validation Using Real-Time RT-PCR

To independently validate these highlighted miRNAs, real-time RT-PCRassays were conducted using total RNA isolated from pulmonaryadenocarcinomas and adjacent normal lung tissues from the describedmurine transgenic lines. As shown, miR-136, miR-376a, miR-31 (FIG. 10)and others (data not shown) were each significantly expressed byreal-time RT-PCR assays using RNA derived from transgenic malignant (T)versus normal transgenic (N) or Tg− lung tissues. Each assay wasconducted at least three independent times with similar results. Resultsobtained were concordant with findings from miRNA microarray experiments(FIG. 10A and data not shown). The results displayed in FIG. 10A frommurine lung tissues are presented because the same miRNAs were alsofrequently over-expressed in human lung cancers versus adjacent normallung tissues, as shown in FIG. 10B.

Real-Time RT-PCR Assays

The miRNA RT-PCR assays were performed using the TaqMan miRNA ReverseTranscription Kit (Applied Biosystems, Foster City, Calif.) and the 7500Fast Real-Time PCR System (Applied Biosystems, Foster City, Calif.) forquantitative miRNA detection, and each miRNA TaqMan PCR probe waspurchased from Applied Biosystems, as described (Liu). The real-timeRT-PCR assays were performed using the 7500 Fast Real-Time PCR Systemfor quantitative mRNA detection and with the iTaq Fast SYBR Greensupermix (BIO-RAD, Hercules, Calif.). TaqMan real-time PCR primer setfrom Applied Biosystems was use. Control RNA primer wa snoRNA-135 formouse and RNU6B for human. Taqman Primers: Human miR-31: 4395390, RNU6B:4373381, Mouse miR-31: 4373331 and SnoRNA-135: 4380912.

Example 9

Over-Expression of miRNAs in Human Lung Cancer

To explore if the identified miRNAs with increased expression in murinelung cancers would also be over-expressed in human lung cancers ascompared to adjacent normal lung tissues, paired human normal-malignantlung tissues were examined from each type of non-small cell lung cancers(NSCLC, adenocarcinoma, AD; squamous cell carcinoma, SC; large cellcarcinoma, LC; and bronchioalveolar carcinoma, BAC) using a previouslydescribed lung tissue bank (Lui, et al and Petty et al). Over-expressionof miR-136, miR-376a and miR-31 was frequently detected in each type ofNSCLC as compared to adjacent normal lung (FIG. 10B). Each of thesehighlighted miRNAs was independently examined in the indicated murineand human lung tissues. The other miRNAs identified as differentiallyover-expressed in murine transgenic lung cancer (FIG. 8) were detectedas over-expressed in only a minority of these NSCLC subtypes (data notshown). Of these differentially over-expressed miRNAs, miR-31 wasselected for in-depth study.

Human Lung Tissues

Paired human normal and malignant lung tissues were obtained afterreview and approval of Dartmouth's Institutional Review Board. Patientidentifying information was not linked to this tissue bank consecutivelyaccrued over 8 years at Dartmouth-Hitchcock Medical Center.

Example 10 Knock-Down of miR-31 in Lung Cancer Cells

The effect of the highlighted miRNAs on lung cancer cell growth wasinvestigated. Each species was independently transiently engineered withincreased or decreased expression in murine lung cancer cell lines (ED-1and ED-2) as well as in the C10 alveolar type II epithelial cell line.Independent over-expression of miR-136, miR-376a and miR-31 in thesecell lines did not appreciably affect murine lung cell growth (data notshown). This was likely due to the high basal levels of these miRNAs inthese respective cell lines that prevented eliciting further effectsfrom engineering even higher expression levels. To confirm this,real-time RT-PCR assays were independently conducted (as describedabove) on these cell lines as well as on murine and human lung cancertissues to assess the relative levels of the miRNAs of interest ascompared to control species (sno-135 for murine cells/tissues and RUN6Bfor human cells/tissues). Findings revealed that miR-31 expressionlevels were higher in nearly all these cell lines examined (cancer andnormal cell lines) than in murine (FIG. 11A) or human (FIG. 11B) lungcancer tissues.

In contrast, knock-down of miR-31 significantly reduced lung cancer cellgrowth as compared to controls, as shown in FIG. 12A upper panel, inwhich miR-31 was independently knocked-down in murine lung cancer celllines (ED-1 and ED-2) as well as in C10 cells. As displayed in FIG. 12A,ED-1 and ED-2 cell growth was suppressed more than 60% (P<0.0001), whileC-10 cell growth was less prominently reduced (P<0.005). Appreciableincrease of apoptosis was not observed by knock-down of miR-31 ascompared to controls (data not shown). Over 90% of cells from each cellline were transfected (data not shown) and miR-31 levels in eachknock-down transfectant was reduced to less than 10% as compared tocontrol transfectants, as confirmed by real-time RT-PCR assays (FIG.12A, lower panel). It was hypothesized that similar effects would beobserved in BEAS-2B human immortalized bronchial epithelial cells versushuman lung cancer cell lines. The same transfection experiments wereindependently conducted in H23 and H226 human lung cancer cell lines aswell as in BEAS-2B cells. A significant (P=0.00019 for H23 and P=0.015for H226) growth suppressive effect was independently caused byknock-down of miR-31 in H23 and H226 cells (FIG. 12A). Intriguingly,proliferation of transfected BEAS-2B cells was not significantlyaffected by engineered miR-31 knock-down, suggesting a differentresponse to loss of miR-31 expression in human immortalized versusmalignant lung epithelial cells. To exclude non-specific transfectioneffects and to examine whether this growth inhibition was reversible,miR-31 was independently knocked-down in ED-1 and ED-2 cells and growthsuppression was examined in these transfectants. Two days after thefirst transfection, pre-miR-31 or an inactive control pre-miR wastransiently over-expressed in these respective cells and growth effectswere examined. As expected, engineered over-expression of miR-31reversed anti-miR-31-mediated growth suppression (FIG. 12B, left panelsfor each respective transfectant). The miR-31 expression levels in theindicated transfectants were determined by real-time RT-PCR assays (FIG.12B, right panels for each respective transfectants).

Cell Lines

The murine lung cancer cell lines (ED-1 and ED-2 cells) were derived asdescribed previously from wild-type cyclin E and proteasome-degradationresistant cyclin E transgenic mice, respectively (Luie et al). The C10murine alveolar type II epithelial cell line and H226, H23, HOP62, H522and A549 human lung cancer cell lines were each purchased from ATCC.BEAS-2B immortalized human bronchial epithelial cells were kindlyprovided by Dr. Curtis C. Harris (National Institutes of Health andNational Cancer Institute, Bethesda, Md.).

Tissue Culture

ED-1, ED-2, H226, H23, HOP62, H522 and A549 cells were each cultured inRPMI 1640 medium with 10% FBS and 1% antibiotic and antimycotic solutionin a humidified incubator at 37° C. in 5% CO2. C10 cells were culturedin CMRL 1066 medium (Life Technologies, Grand Island, N.Y.) with 10%FBS, 2 mM L-glutamine, 100 units/ml penicillin, and 100 μg/mlstreptomycin. BEAS-2B cells were cultured in serum-free LHC-9 medium(Invitrogen, Carlsbad, Calif.).

Transient Transfection

ED-1, ED-2, C10, H23, H226 and BEAS-2B cells were individually platedsubconfluently onto each well of 6-well tissue culture plates or 10 cmdishes 24 hours before transfection. Transient transfection of pre-miRmiRNA precursors and/or anti-miR and control oligonucleotides(anti-miR-neg or pre-miR-neg) (Ambion, Austin, Tex.) at a finalconcentration of 50 nM was accomplished with siPORT NeoFX reagent(Ambion, Austin, Tex.) using previously optimized methods (Lui et al).Logarithmically growing transfectants were harvested for independentimmunoblot, proliferation, apoptosis, and trypan blue viability assays,as described. Over-expression of miR-31 were obtained using pre-miR-31(pre-miR-neg as control) from Applied Biosystems. The chemicallymodified oligonucleotides identified as follows Human Pre-miR-31:AM17100, Mouse pre-miR-31: AM17101 and Pre-miR-neg: AM17110 were used.To knock-down miR-31, anti-miR-31 (anti-miR-neg as control) from AppliedBiosystems were used. Human anti-miR-31: AM17000, Mouse anti-miR-31:AM17001 and Anti-miR-neg: AM17010.

Example 11 Knock-Down of miR-31 Represses In Vivo Tumorigenicity

To examine if this growth inhibitory effect of mrR-31 knock down causesreduced clonal growth of lung cancer cells colony formatin assays wereperformed. Independent knock-down of miR-31 significantly reduced ED-1and ED-2 colony formation assays, as shown in FIG. 13A. The colonynumbers for anti-miR-31 transfectants wereas significantly less than forthe control groups, with P=0.018 for ED-1 cells and P=0.0002 for ED-2cells, respectively. To examine whether the observed repression of lungcancer cell clonal growth was associated with in vivo repression oftumorigenicity, syngeneic FVB mice were tail-vein injected with ED-1cells (see the Materials and Methods) that were transiently transfectedwith anti-miR-31 to achieve knock-down of miR-31. Results were comparedto control transfectants. Twenty-five days after tail-vein injections,lung lesions were scored. As displayed in FIG. 13B, ED-1 cellstransfected with anti-miR-31 produced significantly fewer (P=0.05) lunglesions as compared to ED-1 control transfectants. The knock-down ofmiR-31 by transient transfection persisted for approximately 6 dayswhile the outgrowth of lung lesions occurred typically by 7-15 days postinjection (data not shown).

Proliferation and Colony Formation Assays

The CellTiter-Glo proliferation assay (Promega, Madison, Wis.) was usedalong with previously optimized methods (Lui et al). The colonyformation assay was performed with 2.5×102 ED-1 cells or 5×102 ED-2cells or the indicated transfectants independently plated onto 10 cmtissue culture plates. After 10 days, visible colonies were fixed andstained with Diff-Quik solution (Baxtor, Deerfield, Ill.) and quantifiedusing the Col Count instrument (Oxford Optronix, UK), as described (Luiet al).

In Vivo Tumorigenicity and Statistical Assays

Early passages of ED-1 cells were harvested in PBS supplemented with 10%mouse serum (Invitrogen, Carlsbad, Calif.) and 106 cells of eachtransfectant of this cell line were individually injected into tailveins of each respective FVB syngeneic mouse. In each experimental arm10 control mice tail vein-injected with control transfected ED-1 cellsand 10 mice tail vein-experimental injected with miR-31 (miR-31knock-down ED-1 transfectants) mice were used. With a replicateexperiment, a total of 40 mice were examined. Post-injection (17 days)mice were sacrificed following an Institution Animal Care and UseCommittee (IACUC) approved protocol and harvested lung tissues wereformalin-fixed, paraffin-embedded, sectioned as well as hematoxylin andeosin stained for histopathologic analyses using optimized methods (Maet al). Histopathologic sections were scored for lung tumors by apathologist (H.L.), who was unaware of the treatment arms beinganalyzed. The log transformation of these data was used to eliminate theskewness of counts with the subsequent application of the two-tailt-test for comparison of the number of lung lesions foci in the FVB miceinjected with anti-miR-neg versus anti-miR-31 transfectants. Thedifference was scored as statistically significant if the p-value was0.05 or less. For an alternative statistical analysis, thelikelihood-ratio test was used with the assumption that counts follow aPoisson distribution. Computations were conducted using the statisticalpackage S-Plus 6.1 (Insightful Inc., Seattle, Wash.).

Example 12 Bioinformatic mRNA Targets

Bioinformatic analyses were performed to search for miR-31 target mRNAs.The PicTar, TargetScan 4.0 and online miRNA target prediction programs(http COLON SLASH SLASH cbio DOT mskcc DOTorg/cgi-bin/mirnaviewer/mirnaviewer.pl) were each used to independentlypredict miR-31 targets. It is apparent that each miRNA can affectmultiple targets via distinct mechanisms. Given this, attention focusedon finding candidate tumor suppressive genes that could exert miR-31growth inhibitory effects. Several candidates were highlighted acrosseach bioinformatic program used. These were LATS2, PPP2R2A, Frizzledhomolog 3 (FZD3), Sprouty-related, EVH1 domain containing 1 (SPRED1),Sprouty homolog 4 (SPRY4), AXIN1 up-regulated 1 (AXUD1), and DICER1. ThemiR-31 targets sought were those that were repressed by forced miR-31over-expression and augmented by its engineered knock-down. Among thesecandidates, only LATS2 and PPP2R2A satisfied this criterion, as shown inFIGS. 14A and 14B, respectively. All six cell lines (ED-1, ED-1, C10,H23, H226 and BEAS-2B) were independently examined in these analyses andeach of them exhibited concordant results.

LATS2, human large tumor suppressor homolog 2 (also known as KPM), is amember of LATS tumor suppressor family. PPP2R2A, also known as proteinphosphatase 2A B55 subunit act as a tumor suppressor by induction ofapoptosis.

To functionally determine whether these species are targets of miR-31,LATS2 and PPP2R2A were individually knocked-down by siRNAs in ED-1 cells48 hours after the initial transfection that conferred miR-31 repression(as described above in example 10). As expected, siRNA knock-down ofLATS2 and PPP2R2A each antagonized the growth suppression caused byengineered miR-31 repression (FIG. 15A). Two independently designedsiRNAs were used to target LATS2 and PPP2R2A and each experiment wasconducted in triplicate at least three independent times. Compared withcontrol siRNA, LATS2 siRNAs and PPP2R2A siRNAs repressed the desiredtarget mRNA levels to 5-6% of basal levels as examined by real-timeRT-PCR assays (FIG. 15B). These results demonstrated that repression ofLATS2 and PPP2R2A expression antagonized growth suppression caused bymiR-31 knock-down and functionally validated them as miR-31 targets.Similar results were independently obtained in experiments with ED-2cells, as shown in FIGS. 15C and 15D.

The siRNA transfections were conducted following the same procedure asfor a pre-miR and used at a final concentration of 25 nM (see above inrelation to example 10). The Silencer Select Pre-designed siRNAs werepurchased from Applied Biosystems (Foster City, Calif.) and LATS2 siRNAs(s78350 and s78351), PPP2R2A siRNAs (s90396 and s90395) and a negativecontrol siRNA (4390843) wereas also used in these experiments.

Example 13 LATS2 and PPP2R2A Expression in Lung Cancers

Since miR-31 was over-expressed in both murine cyclin E transgenic lungcancer models and in human lung cancer tissues, we sought to determinewhether LATS2 and PPP2R2A were each basally repressed in these lungcancers relative to adjacent normal lung tissues. Real-time RT-PCRassays were conducted on murine cyclin E transgenic lung adenocarcinomasand adjacent normal lung tissues, as well as in previously studiedpaired human normal-malignant lung tissues (Lui et al). Both LATS2 andPPP2R2A were each basally repressed in these murine cyclin E transgeniclung cancers, with mRNA levels substantially reduced as compared toadjacent normal lung tissues, as in FIGS. 16A and 16B. As expected,LATS2 and PPP2R2A expression was also repressed in all examined humanlung cancers relative to adjacent normal tissues, as shown in FIGS. 16Cand 16D. These results underscore that concordant expression profileswere found for miR-31 and its target mRNAs in both murine and human lungcancers.

Real-Time RT-PCR Assays were beformed as described above in relation toexample 8 using the below primers for amplification.

human GAPDH: SEQ ID NO 38, 5′-ATGGGGAAGGTGAAGGTCG-3′ (forward) andSEQ ID NO 39, 5′-GGGGTCATTGATGGCAACAATA-3′ (reverse); human PPP2R2A:SEQ ID NO 40, 5′-TCGGATGTAAAATTCAGCCA-3′ (forward) andSEQ ID NO 41, 5′-CATGCACCTGGTATGTTTCC-3′ (reverse); human LATS2:SEQ ID NO 42, 5′-CAGATTCAGACCTCTCCCGT-3′ (forward) andSEQ ID NO 43, 5′-CTTAAAGGCGTATGGCGAGT-3′ (reverse); mouse GAPDH:SEQ ID NO 44, 5′-AGGTCGGTGTGAACGGATTTG-3′ (forward) andSEQ ID NO 45, 5′-TGTAGACCATGTAGTTGAGGTCA-3′ (reverse); mouse LATS2:SEQ ID NO 46, 5′-AGCAGATTGTGCGAGTCATC-3′ (forward) andSEQ ID NO 47, 5′-GTGGTAGGATGGGAGTGCTT-3′ (reverse); mouse PPP2R2A:SEQ ID NO 48, 5′-TAAGAGAGCGGTCCATTGTG-3′ (forward) andSEQ ID NO 49, 5′-ACAGCTTTCTCCATGAGGCT-3′ (reverse)

REFERENCES

-   Abelson, J. F., Kwan, K. Y., O'Roak, B. J., Baek, D. Y.,    Stillman, A. A., Morgan, T. M., Mathews, C. A., Pauls, D. L.,    Rasin, M. R., Gunel, M., Davis, N. R., Ercan-Sencicek, A. G.,    Guez, D. H., Spertus, J. A., Leckman, J. F., Dure, L. S. 4th,    Kurlan, R, Singer, H. S., Gilbert, D. L., Farhi, A., Louvi, A.,    Lifton, R. P., Sestan, N., State, M. W. 2005. Sequence variants in    SLITRK1 are associated with Tourette's syndrome. Science 310:    317-20.-   Aboobaker, A. A., Tomancak, P., Patel, N., Rubin, G. M.,    Lai, E. C. 2005. Drosophila microRNAs exhibit diverse spatial    expression patterns during embryonic development.-   Proc Natl Acad Sci USA. 102:18017-22.-   't Veer, L. J., Dai, H., van de Vijver, M. J., He, Y. D., Hart, A.    A., Mao, M., Peterse, H. L., van der, K. K., Marton, M. J.,    Witteveen, A. T., Schreiber, G. J., Kerkhoven, R. M., Roberts, C.,    Linsley, P. S., Bernards, R., and Friend, S. H. (2002). Gene    expression profiling predicts clinical outcome of breast cancer.    Nature 415, 530-536.-   Altuvia, Y., Landgraf, P., Lithwick, G., Elefant, N., Pfeffer, S.,    Aravin, A., Brownstein, M. J., Tuschl, T., Margalit, H. 2005.    Clustering and conservation patterns of human microRNAs. Nucleic    Acids Res. 33: 2697-706.-   Barad, O., Meiri, E., Avniel, A., Aharonov, A., Barzilai, A.,    Bentwich, I., Einav, U., Gilad, S., Hurban, P., Karov, Y.,    Lobenhofer, E. K., Sharon, E., Shiboleth, Y. M., Shtutman, M.,    Bentwich, Z., and Einat, P. 2004. MicroRNA expression detected    microarrays: System establishment profiling in human tissues. Genome    Research 14: 2486-2494.-   Bartel, D. P. 2004. MicroRNAs: Genomics, biogenesis, mechanism and    function. Cell 116: 281-297.-   Bentwich, I., Avniel, A., Karov, Y., Aharonov, R., Gilad, S., Barad,    O., Barzilai, A., Einat, P., Einav, U., Meiri, E., Sharon, E.,    Spector, Y. and Bentwich, Z. 2005. Identification of hundreds of    conserved and nonconserved human microRNAs. Nat. Genet. 37: 766-770.-   Berezikov, E., Guryev, V., van de Belt, J., Wienholds, E.,    Plasterk, R. H. A. and Cuppen, E. 2005. Phylogenetic shadowing and    computational identification of human microRNA genes. Cell 120:    21-24-   Boehm, M., Slack, F. 2005. A developmental timing microRNA and its    target regulate life span in C. elegans. Science. 310:1954-7.-   Brennecke, J., Hipfner, D. R., Stark, A., Russel, R. B. and    Cohen S. 2003. bantam encodes a developmentally regulated microRNA    that controls cell proliferation and regulates the proapoptotic gene    hid in Drosophila. Cell 113: 25-36.-   Brennecke, J., Stark, A., Russel, R. B. and Cohen S 2005. Principles    of MicroRNA-target recognition. PLoS Biology 3: e85-   Calin, G. A., Sevignani, C., Dumitru, C. D., Hyslop, T., Noch, E.,    Yendamuri, S., Shimizu, M., Rattan, S., Bullrich, F., Negrini, M.    and Croce, C. M. 2004. Human microRNA genes are frequently located    at fragile sites and genomic regions involved in cancers. Proc.    Natl. Acad. Sci. U.S.A. 101: 2999-3004.-   Calin, G. A., Dumitru, C. D., Shimizu, M., Bichi, R., Zupo, S.,    Noch, E., Aldler, H., Rattan, S., Keating, M., Rai, K., Rassenti,    L., Kipps, T., Negrini, M., Bullrich, F. and Croce, C. M. 2002.    Frequent deletions and down-regulation of microRNA genes miR15 and    miR16 at 13q14 in chronic lymphocytic leukemia. Proc. Natl. Acad.    Sci. USA 99: 15524-15529.-   Calin, G. A., Ferracin, M., Cimmino, A., Di Leva, G., Shimizu, M.,    Wojcik, S. E., Iorio, M. V., Visone, R.; Sever, N. I., Fabbri, M.,    Iuliano, R., Palumbo, T., Pichiorri, F., Roldo, C., Garzon, R.,    Sevignani, C., Rassenti, L., Alder, H., Volinia, S., Liu, C. G.,    Kipps, T. J., Negrini, M., Croce, C. M. 2005. A MicroRNA signature    associated with prognosis and progression in chronic lymphocytic    leukemia. N. Engl. J. Med. 353:1793-801-   Chan, J. A., Krichevsky, A. M., Kosik, K. S. 2005. MicroRNA-21 is an    antiapoptotic factor in human glioblastoma cells. Cancer Res.    65:6029-33.-   Chen, C. Z., Li, L., Lodish, H. F. and Bartel, D. P. 2004. MicroRNAs    modulate hematopoietic lineage differentiation. Science 303: 83-86.-   Chen, X. 2004. A MicroRNA as a translational repressor of APETALA2    in Arabidopsis flower development. Science 203: 2022-2025;-   Chen, J. F., Mandel, E. M., Thomson, J. M., Wu, Q., Callis, T. E.,    Hammond, S. M., Conlon, F. L., Wang, D. Z. 2005. The role of    microRNA-1 and microRNA-133 in skeletal muscle proliferation and    differentiation. Nat Genet. December 25, advance online publication.-   Colozza, M., Cardoso, F., Sotiriou, C., Larsimont, D., and    Piccart, M. J. (2005). Bringing molecular prognosis and prediction    to the clinic. Clin. Breast Cancer. 6, 61-76.-   E is, P. S., Tam, W., Sun, L., Chadburn, A., Li, Z., Gomez, M. F.,    Lund, E., Dahlberg, J. E. 2005. Accumulation of miR-155 and BIC RNA    in human B cell lymphomas. Proc Natl Acad Sci USA. 102: 3627-32.-   Farh, K. K., Grimson, A., Jan, C., Lewis, B. P., Johnston, W. K.,    Lim, L. P., Burge, C. B., Bartel, D. P. 2005. The widespread impact    of mammalian MicroRNAs on mRNA repression and evolution. Science.    310:1817-21.-   Giraldez, A. J., Cinalli, R. M., Glasner, M. E., Enright, A. J.,    Thomson, J. M., Baskerville, S., Hammond, S. M., Bartel, D. P. and    Schier, A. F. 2005. MicroRNAs regulate brain morphogenesis in    zebrafish. Science 308: 833-838.-   Griffiths-Jones, S. 2004. The microRNA Registry. Nucleic Acids Res.    32: D109-D111.-   Griffiths-Jones, S., Grocock, R. J., van Dongen, S., Bateman, A.,    Enright, A. J. 2006. miR-Base: microRNA sequences, targets and gene    nomenclature. Nucleic Acids Res. 34: D140-4-   He, L., Thomson, J. M., Hemann, M. T., Hernando-Monge, E., Mu, D.,    Goodson, S., Powers, S., Cordon-Cardo, C., Lowe, S. W.,    Hannon, G. J. and Hammond, S. M. 2005. A microRNA polycistron as a    potential human oncogene. Nature 435: 828-833.-   Hornstein, E., Mansfield, J. H., Yekta, S., Hu, J. K., Harfe, B. D.,    McManus, M. T., Baskerville. S., Bartel, D. P., Tabin, C. J. 2005.    The microRNA miR-196 acts upstream of Hoxb8 and Shh in limb    development. Nature 438: 671-4.-   Huber, W., Heydebreck, A., Sültmann, H., Poustka, A., and    Vingron, M. (2002) Variance stabilization applied to microarray data    calibration and to quantification of differential expression.    Bioinformatics 18: 96-104.-   Hutvagner, G., McLachlan, J., Pasquinelli, A. E., Balint, E.,    Tuschl, T. and Zamore, P. D. 2001. A cellular function for the    RNA-interference enzyme Dicer in the maturation of the let-7 small    temporal RNA. Science 293: 834-838.-   Hutvagner, G., Simard, M. J., Mello, C. C. and Zamore, P. D. 2004.    Sequence-specific inhibition of small RNA function. PLoS Biology 2:    1-11.-   Jin, P., Alisch, R. S., Warren, S. T. 2004. RNA and microRNAs in    fragile X mental retardation. Nat Cell Biol. 6:1048-53.-   Johnson, S. M., Grosshans, H., Shingara, J., Byrom, M., Jarvis, R.,    Cheng, A., Labourier, E., Reinert, K. L., Brown, D. and    Slack, F. J. 2005. RAS is regulated by the let-7 microRNA family.    Cell 120: 635-647.-   Johnston, R. J. and Hobert, O. 2003. A microRNA controlling    left/right neuronal asymmetry in Caenorhabditis elegans. Nature 426:    845-849.-   Jopling, C. L., Yi, M., Lancaster, A. M., Lemon, S. M.,    Sarnow, P. 2005. Modulation of hepatitis C virus RNA abundance by a    liver-specific MicroRNA. Science 309:1577-81.-   Ketting, R. F., Fischer, S. E., Bernstein, E., Sijen, T.,    Hannon, G. J. and Plasterk, R. H. 2001. Dicer functions in RNA    interference and in synthesis of small RNA involved in    develop-mental timing in C. elegans. Genes Dev. 15: 2654-2659.-   Kim, J., Krichevsky, A., Grad, Y., Hayes, G. D., Kosik, K. S.,    Church, G. M., Ruvkun, G. 2004. Identification of many microRNAs    that copurify with polyribosomes in mammalian neurons. Proc Natl    Acad Sci USA. 101: 360-5.-   Krek, A., Grun, D., Poy, M. N., Wolf, R., Rosenberg, L., Epstein, E.    J., MacMenamin, P., da Piedade, I., Gunsalus, K. C., Stoffel, M. and    Rajewsky, N. 2005. Combinatorial microRNA target predictions. Nat.    Genet. 37: 495-500.-   Krichevsky, A. M., King, K. S., Donahue, C. P., Khrapko, K. and    Kosik, K. S. 2003. A microRNA array reveals extensive regulation of    microRNAs during brain development. RNA 9: 1274-1281.-   Krützfeldt, J., Rajewsky, N., Braich, R., Rajeev, K. G., Tuschl, T.,    Manoharan, M. and Stoffel, M. 2005. Silencing of microRNAs in vivo    with ‘antagomirs’. Nature 438: 685-9.-   Kwon, C., Han, Z., Olson, E. N., Srivastava, D. 2005. MicroRNA1    influences cardiac differentiation in Drosophila and regulates Notch    signaling. Proc Natl Acad Sci USA. 102: 18986-91.-   Lagos-Quintana, M., Rauhut, R., Lendeckel, W. and Tuschl, T. 2001.    Identification of novel genes coding for small expressed RNAs.    Science 294: 853-858.-   Landthaler, M., Yalcin, A. and Tuschl, T. 2004. The human DiGeorge    syndrome critical region gene 8 and its D. melanogaster homolog are    required for miRNA biogenesis. Curr. Biol. 14: 2162-2167.-   Leaman, D., Chen, P. Y., Fak, J., Yalcin, A., Pearce, M.,    Unnerstall, U., Marks, D. S., Sander, C., Tuschl, T., Gaul, U. 2005.    Antisense-mediated depletion reveals essential and specific    functions of microRNAs in Drosophila development. Cell 121:    1097-108.-   Lee, R. C. and Ambros, V. 2001. An extensive class of small RNAs in    Caenorhabditis elegans. Science 294: 862-864.-   Lee, R. C., Feinbaum, R. L. and Ambros, V. 1993. The C. elegans    heterochronic gene lin-4 encodes small RNAs with antisense    complementarity to lin-14. Cell 75: 843-854.-   Lee, Y., Ahn, C., Han, J., Choi, H., Kim, J., Yim, J., Lee, J.,    Provost, P., Radmark, O., Kim, S, and Kim, V. N. 2003. The nuclear    RNase III Drosha initiates microRNA processing. Nature 425: 415-419.-   Lewis, B. P., Burge, C. B., Bartel, D. P. 2005. Conserved seed    pairing, often flanked by adenosines, indicates that thousands of    human genes are microRNA targets. Cell 120: 15-20.-   Li, X. and Carthew, R. W. 2005. A microRNA mediates EGF receptor    signaling and pro-motes photoreceptor differentiation in the    drosophila eye. Cell 123: 1267-77.-   Liu, X., Sempere, L. F., Galimberti, F., Freemantle, S. J., Black,    C., Dragnev, K. H., Ma, Y., Fiering, S., Memoli, V., Li, H., et    al. 2009. Uncovering growth-suppressive MicroRNAs in lung cancer.    Clin Cancer Res 15:1177-1183.-   Lim, L. P., Lau, N. C., Garrett-Engele, P., Grimson, A.,    Schelter, J. M., Castle, J., Bartel, D. P., Linsley, P. S., and    Johnson, J. M. 2005. Microarray analysis shows that some microRNAs    downregulate large numbers of target mRNAs. Nature 433: 769-773.-   Liu, C-G., Calin, G. A., Meloon, B., Gamliel, N., Sevignani, G.,    Ferracin, M., Dumitru, C. M., Shimizu, M., Zupo, S., Dono, M.,    Alder, H., Bullrich, F., Negrini, M. and Croce, C. M. 2004. An    oligonucleotide microchip for genome-wide microRNA profiling in    human and mouse tissues. Proc. Natl. Acad. Sci, U.S.A.    101:9740-9744,-   Lu. J., Getz, G., Miska, E. A., Alvarez-Saavedra, E. A., Lamb, J.,    Peck, D., Sweet-Cordero, A., Ebert, B. L., Mak, R. H., Ferrando, A.    A., Downing, J. R., Jacks, T., Horvitz, H. R., and    Golub, T. R. 2005. MicroRNA expression profiles classify human    cancers. Nature 435: 834-838.-   Ludwig, J. A. and Weinstein, J. N. (2005). Biomarkers in cancer    staging, prognosis and treatment selection. Nat. Rev. Cancer. 5,    845-856.-   Ma, Y., Fiering, S., Black, C., Liu, X., Yuan, Z., Memoli, V. A.,    Robbins, D. J., Bentley, H. A., Tsongalis, G. J., Demidenko, E., et    al. 2007. Transgenic cyclin E triggers dysplasia and multiple    pulmonary adenocarcinomas. Proc Natl Acad Sci USA 104:4089-4094.-   Michael, M. Z., SM, O. C., van Hoist Pellekaan, N. G., Young, G. P.    and James, R. J. 2003. Reduced accumulation of specific microRNAs in    colourectal neoplasia. Mol. Cancer. Res. 1: 882-891.-   Nelson, P., Kiriakidou, M., Sharma, A., Maniataki, E. and    Mourelatos, Z. 2003. The microRNA world: small is mighty. TIBS 28:    534-540.-   Petty, W. J., Li, N., Biddle, A., Bounds, R., Nitkin, C., Ma, Y.,    Dragnev, K. H., Freemantle, S. J., and Dmitrovsky, E. 2005. A novel    retinoic acid receptor beta isoform and retinoid resistance in lung    carcinogenesis. J Natl Cancer Inst 97:1645-1651.-   Ocana, A., Cruz, J. J., and Pandiella, A. (2006). Trastuzumab and    antiestrogen therapy: focus on mechanisms of action and resistance.    Am. J. Clin. Oncol. 29, 90-95.-   Paushkin, S., Gubitz, A. K., Massenet, S, and Dreyfuss, G. 2002. The    SMN complex, an assemblyosome of ribonucleoproteins. Curr. Opin.    Cell Biol. 14: 305-312.-   Perou, C. M., Jeffrey, S. S., van de, R. M., Rees, C. A., Eisen, M.    B., Ross, D. T., Pergamenschikov, A., Williams, C. F., Zhu, S. X.,    Lee, J. C., Lashkari, D., Shalon, D., Brown, P. O., and Botstein, D.    (1999). Distinctive gene expression patterns in human mammary    epithelial cells and breast cancers. Proc. Natl. Acad. Sci. U.S. A    96, 9212-9217.-   Poy, M. N., Eliasson, L., Krutzfeldt, J., Kuwajima, S., Ma, X.,    Macdonald, P. E., Pfeffer, S., Tuschl, T., Rajewsky, N., Rorsman, P.    and Stoffel, M. (2004) A pancreatic islet-specific microRNA    regulates insulin secretion. Nature 432: 226-230.-   Reinhart, B. J., Slack, F. J., Basson, M., Bettinger, J. C.,    Pasquinelli, T., Rougvie, A. E., Horvitz, H. R., and    Ruvkun, G. 2000. The 21 nucleotide let-7 RNA regulates developmental    timing in Caenorhabditis elegans. Nature 403: 901-906.-   Reinhart, B. J., Weinstein, E. G., Rhoades, M. W., Bartel, B., and    Bartel, D. P. 2002. MicroRNAs in plants. Genes Dev. 16: 1616-1626.-   Rodriguez, A., Griffiths-Jones, S., Ashurst, J. L., Bradley, A.    Related 2004. Identification of mammalian microRNA host genes and    transcription units. Genome Res. 10A: 1902-10.-   Saeed, A. I., Sharov, V., White, J., Li, J., Liang, W., Bhagabati,    N., Braisted, J., Klapa, M., Currier, T., Thiagarajan, M., Sturn,    A., Snuffin, M., Rezantsev, A., Popov, D., Ryltsov, A., Kostukovich,    E., Borisovsky, I., Liu, Z., Vinsavich, A., Trush, V., and    Quackenbush, J. (2003).-   TM4: a free, open-source system for microarray data management and    analysis. Biotechniques 34, 374-378.-   Seitz, H., Royo, H., Bortolin, M. L., Lin, S. P., Ferguson-Smith, A.    C., Cavaille, J. 2004. A large imprinted microRNA gene cluster at    the mouse Dlk1-Gtl2 domain. Genome Res. 9: 1741-8.-   Sempere, L. F., Christensen, M., Silahtaroglu, A., Bak, M.,    Heath, C. V., Schwartz, G., Wells, W., Kauppinen, S., and    Cole, C. N. 2007. Altered MicroRNA expression confined to specific    epithelial cell subpopulations in breast cancer. Cancer Res    67:11612-11620.-   Smirnova, L., Grafe, A., Seiler, A., Schumacher, S., Nitsch, R. and    Wulczyn, F. G. 2005. Regulation of miRNA expression during neural    cell specification. Eur J Neurosci. 21: 1469-77.-   Sokol, N. S., Ambros, V. 2005. Mesodermally expressed Drosophila    microRNA-1 is regulated by Twist and is required in muscles during    larval growth. Genes Dev. 19: 2343-54.-   Sorlie, T. (2004). Molecular portraits of breast cancer: tumour    subtypes as distinct disease entities. Eur. J. Cancer. 40,    2667-2675.-   Sorlie, T., Perou, C. M., Tibshirani, R., Aas, T., Geisler, S.,    Johnsen, H., Hastie, T., Eisen, M. B., van de, R. M., Jeffrey, S.    S., Thorsen, T., Quist, H., Matese, J. C., Brown, P. O., Botstein,    D., Eystein, L. P., and Borresen-Dale, A. L. (2001). Gene expression    patterns of breast carcinomas distinguish tumour subclasses with    clinical implications. Proc. Natl. Acad. Sci. U.S.A. 98,    10869-10874.-   Sorlie, T., Tibshirani, R., Parker, J., Hastie, T., Marron, J. S.,    Nobel, A., Deng, S., Johnsen, H., Pesich, R., Geisler, S., Demeter,    J., Perou, C. M., Lonning, P. E., Brown, P. O., Borresen-Dale, A.    L., and Botstein, D. (2003). Repeated observation of breast tumour    subtypes in independent gene expression data sets. Proc. Natl. Acad.    Sci. U.S. A 100, 8418-8423.-   Stark, A., Brennecke, J., Bushati, N., Russell, R. B.,    Cohen, S. M. 2005. Animal MicroRNAs confer robustness to gene    expression and have a significant impact on 3′UTR evolution. Cell    123: 1133-46.-   Wienholds, E., Kloosterman, W. P., Miska, E., Alvarez-Saavedra, E.,    Berezikov, E., de Bruijn, E., Horvitz, H. R., Kauppinen, S, and    Plasterk, R. H. A. 2005. MicroRNA expression in zebrafish embryonic    development. Science 309: 310-311.-   Xie, X., Lu, J., Kulbokas, E. J., Golub, T. R., Mootha, V.,    Lindblad-Toh, K., Lander, E. S. and Kellis, M. 2005. Systematic    discovery of regulatory motifs in human promoters and 30 UTRs by    comparison of several mammals. Nature 434: 338-345.-   Yekta, S., Shih, I-s and Bartel, D. 2004. microRNA-Directed Cleavage    of HOXB8 mRNA. Science 304: 594-596.-   Zhao, Y., Samal, E. and Srivastava, D. 2005. Serum response factor    regulates a muscle-specific microRNA that targets Hand2 during    cardiogenesis. Nature 436: 214-220.

1-20. (canceled)
 21. A kit for diagnosing lung cancer comprising acombination of nucleotide molecules that hybridize under stringentconditions to nucleotide molecules selected from the group of SEQ IDNO:1, 3, 4, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and
 29. 22. A kitfor diagnosing breast cancer comprising a combination of nucleotidemolecules that hybridize under stringent conditions to nucleotidemolecules selected from the group of SEQ ID NO:1, 5, 6, 7, 8, 9, 13, 14,15, 16, 17, and
 18. 23. A pharmaceutical composition for treating lungcancer comprising a) an oligonucleotide that i) down-regulates theover-expression of the miRNA or pre-miRNA of SEQ ID NO:4, 26, 27, or 28in lung cancer, or ii) increases the levels of the miRNA or pre-miRNA ofSEQ ID NO:1, 3, 19, 20, 21, 22, 23, 24, or 29 in lung cancer; and b) asecond active ingredient for prophylactic or therapeutic treatment oflung cancer.
 24. A pharmaceutical composition for treating breast cancercomprising a) an oligonucleotide that i) down-regulates theover-expression of the miRNA or pre-miRNA of SEQ ID NO:4, 5, 6, 7, 8,12, 13, 14, 15, 16, 17, or 18 in breast cancer, or ii) increases thelevels of the miRNA or pre-miRNA of SEQ ID NO:1, 2, 3, 9, 10, or 11 inbreast cancer; and b) a second active ingredient for prophylactic ortherapeutic treatment of breast cancer.
 25. A method for detecting,classifying, diagnosing or prognosing lung cancer comprising: a)obtaining at least one lung sample from an individual, b) detecting thepresence or absence of expression and/or the expression level of atleast one nucleic acid molecule in cells of the sample, said nucleicacid molecule comprising: i) a nucleotide sequence selected from thegroup consisting of SEQ ID NOs: 1, 3, 4, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, and 29 and/or, ii) a nucleotide sequence which is complementaryto i) and/or, iii) a nucleotide sequence which is a fragment of or ii)and/or, iv) a nucleotide sequence which has an identity of at least 80%to a sequence of i), ii) or iii) and/or, v) a nucleotide sequence whichhybridizes under stringent conditions to a sequence of i), ii), iii) oriv), for the detection of lung cancer in said individual.
 26. A methodfor detecting, classifying, diagnosing or prognosing breast cancercomprising: a) obtaining at least one breast sample from an individual,b) detecting the presence or absence of expression and/or the expressionlevel of at least one nucleic acid molecule in cells of the sample, saidnucleic acid molecule comprising: i) a nucleotide sequence selected fromthe group consisting of SEQ ID NO:5, 6, 7, 8, 9, 13, 14, 15, 16, and 18and/or, ii) a nucleotide sequence which is complementary to i) and/or,iii) a nucleotide sequence which is a fragment of i) or ii) and/or, iv)a nucleotide sequence which has an identity of at least 80% to asequence of i), ii) or iii) and/or, v) a nucleotide sequence whichhybridizes under stringent conditions to a sequence of i), ii), iii) oriv), for the detection of lung cancer in said individual.
 27. A methodfor treating, preventing or reducing the risk of lung cancer associatedwith aberrant expression of a pre-miRNA and/or miRNA in a cell, tissueor animal comprising contacting the cell, tissue or animal with thepharmaceutical composition of claim
 23. 28. The method of claim 27,wherein the oligonucleotide is administered by pulmonary administration.29. A method for treating, preventing or reducing the risk of breastcancer associated with aberrant expression of a pre-miRNA and/or miRNAin a cell, tissue or animal comprising contacting the cell, tissue oranimal with the pharmaceutical composition of claim 24.